WO1996028558A9

WO1996028558A9 - Thermitase variants having decreased adsorption and increased hydrolysis

Info

Publication number: WO1996028558A9
Application number: PCT/US1996/003009
Authority: WO
Filing date: 1996-03-06
Publication date: 1997-01-16

Abstract

The present invention relates to Thermitase variants having a modified amino acid sequence of wild-type Thermitase amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises different amino acids than that occurring in wild-type Thermitase (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the Thermitase variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Thermitase. The present invention also relates to DNA sequences encoding such Thermitase variants. The present invention also relates to compositions comprising such Thermitase variants for cleaning a variety of surfaces.

Description

THERMITASE VARIANTS HAVING DECREASED ADSORPTION AND INCREASED HYDROLYSIS

TECHNICAL FIELD

The present invention relates to novel enzyme variants useful in a variety of cleaning compositions, and DNA sequences encoding such enzyme variants

BACKGROUND

Enzymes make up the largest class of naturally occurring proteins Each class of enzyme generally catalyzes (accelerates a reaction without being consumed) a different kind of chemical reaction One class of enzymes known as proteases, are known for their ability to hydrolyze (break down a compound into two or more simpler compounds with the uptake of the H and OH parts of a water molecule on either side of the chemical bond cleaved) other proteins This ability to hydrolyze proteins has been taken advantage of by incorporating naturally occurring and protein engineered proteases as an additive to laundry detergent preparations Many stains on clothes are proteinaceous and wide-specificity proteases can substantially improve removal of such stains

Unfortunately, the efficacy level of these proteins in their natural, bacterial environment, frequently does not translate into the relatively unnatural wash environment Specifically, protease characteristics such as thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme

The ammo acid sequence of the protease determines the characteristics of the protease A change of the ammo acid sequence of the protease may alter the properties of the enzyme to varying degrees, or may even inactivate the enzyme, depending upon the location, nature and/or magnitude of the change in the ammo acid sequence Several approaches have been taken to alter the wild-type ammo acid sequence of proteases in an attempt to improve their properties, with the goal of increasing the efficacy of the protease in the wash environment These approaches include altering the ammo acid sequence to enhance thermal stability and to improve oxidation stability under quite diverse conditions

Despite the variety of approaches described in the art, there is a continuing need for new effective variants of proteases useful for cleaning a variety of surfaces

Obiects of the Present Invention

It is an object of the present invention to provide Thermitase enzyme variants having improved hydrolysis versus the wild-type of the enzyme

It is also an object of the present invention to provide cleaning compositions comprising these subtilism enzyme variants

SUMMARY

The present invention relates to Thermitase variants having a modified ammo acid sequence of wild-type Thermitase ammo acid sequence the wild-type ammo acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region and a fifth loop region, wherein the modified ammo acid sequence comprises different ammo acids than that occurring in wild-type Thermitase (i e , substitution) at specifically identified positions in one or more of the loop regions whereby the Thermitase variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Thermitase The present invention also relates to DNA sequences encoding such Thermitase variants The present invention also relates to compositions comprising such Thermitase variants for cleaning a variety of surfaces

DESCRIPTION I Thermitase Variants

This invention pertains to subtilism enzymes, in particular Thermitase, that have been modified by mutating the various nucleotide sequences that code for the enzyme, thereby modifying the am o acid sequence of the enzyme The modified subtilism enzymes (hereinafter, "Thermitase variants") of the present invention have decreased adsorption to and increased hydrolysis of an insoluble substrate as compared to the wild-type subtilism The present invention also pertains to DNA sequences encoding for such Thermitase variants

The subtilism enzymes of this invention belong to a class of enzymes known as proteases A protease is a catalyst for the cleavage of peptide bonds One type of protease is a serine protease A serine protease is distinguished by the fact that there is an essential serine residue at the active site

The observation that an enzyme's rate of hydrolysis of soluble substrates increases with enzyme concentration is well documented It would tnerefore seem plausible that for surface bound substrates such as is encountered in many cleaning applications the rate of hydrolysis would increase with increasing surface concentration This has been shown to be the case (Brode, P F III and D S Rauch. LANGMUIR, "Subtilism BPN' Activity on an Immobilized Substrate", Vol 8, pp 1325-1329 (1992)) In fact a linear dependence of rate upon surface concentration was found for insoluble substrates when the surface concentration of the enzyme was varied (Rubmgh D N and M D Bauer "Catalysis of Hydrolysis by Proteases at the Protein-Solution Interface " in POLYMER SOLUTIONS, BLENDS AND INTERFACES Ed by I Noda and D N Rubmgh Elsevier, p 464 (1992)) Surprisingly when seeking to apply this principle in the search for variant proteases which give better cleaning performance, we did not find that enzymes which adsorb more give better performance In fact we surprisingly determined the opposite to be the case decreased adsorption by an enzyme to a substrate resulted in increased hydrolysis of the substrate (i e , better cleaning performance)

While not wishing to be bound by theory, it is believed that improved performance when comparing one variant to another, is a result of the fact that enzymes which adsorb less are also less tightly bound and therefore more highly mobile on the surface from which the insoluble protein substrate is to be removed At comparable enzyme solution concentrations this increased mobility is sufficient to outweigh any advantage that is conferred by delivering a higher concentration of enzyme to the surface

The mutations described herein are designed to change (i.e decrease) the adsorption of the enzyme to surface-bound soils in Thermitase. certain ammo acids form exterior loops on the enzyme molecule For purposes of discussion, these loops shall be referred to as first, second, third, fourth and fifth loop regions Specifically, positions 66- 73 form the first loop region; positions 103-115 form the second loop region, positions 134-141 form the third loop region, positions 162-171 form the fourth loop region, positions 191 -195 form the fifth loop region, and positions 204-224 form the sixth loop region (position numbering analogous to positions in the ammo acid sequence for wild-type subtilism Thermitase (SEQ ID NO:1 )) It is believed that these loop regions play a significant role in the adsorption of the enzyme molecule to a surface-bound peptide, and specific mutations in one or more of these loop regions will have a significant effect on this adsorption While not wishing to be bound by theory, it is believed that the loop regions are important to the adsorption of the Thermitase molecule for at least two reasons First, the ammo acids which comprise the loop regions can make close contacts with any surfaces to which the molecule is exposed Second, the proximity of the loop regions to the active-site and binding pocket of the Thermitase molecule gives them a role in the catalytically productive adsorption of the enzyme to surface-bound substrates (peptides/protem soils)

As used herein, "variant" means an enzyme having an am o acid sequence which differs from that of wild-type

As used herein, "mutant Thermitase DNA" means a DNA sequence coding for a Thermitase variant

As used herein, "wild-type Thermitase" refers to an enzyme represented by SEQ ID NO 1 The ammo acid sequence for Thermitase is further described by Meloun, B , Baudys, M , Kostka, V , Hausdorf, G , Frommel, C , and Hohne, W E , FEBS LETT , Vol 183, pp 195-200 (1985), incorporated herein by reference

As used herein, the term "Thermitase wild-type am o acid sequence" encompasses SEQ ID NO 1 as well as SEQ ID NO 1 having modifications to the ammo acid sequence other than at any of positions 66, 67, 68, 69, 70, 72, 73, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 134, 135, 136, 137, 138, 139, 140, 141 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 191 , 192,193,194, 195, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223 or 224

As used herein, "more hydrophilic ammo acid" refers to any other am o acid having greater hydrophilicity than a subject ammo acid with reference to the hydrophilicity table below The following hydrophilicity table (Table 1 ) lists ammo acids in descending order of increasing hydrophilicity (see Hopp, T P , and Woods, K R., "Prediction of Protein Antigenic Determinants from Am o Acid Sequences", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Vol 78, pp 3824-3828, 1981 , incorporated herein by reference) TABLE 1

Ammo Acid Hydrophilicity Value

Table 1 also indicates which ammo acids carry a charge (this characteristic being based on a pH of from about 8-9) The positively charged am o acids are Arg and Lys, the negatively charged ammo acids are Glu and Asp, and the remaining am o acids are neutral In a preferred embodiment of the present invention, the substituting ammo acid is either neutral or negatively charged, more preferably negatively charged (i e , Glu or Asp)

Therefore, for example, the statement "substitute Gin with an equally or more hydrophilic am o acid which is neutral or has a negative charge" means Gin would be substituted with Asn (which is equally hydrophilic to Gin), or Ser, Glu or Asp (which are more hydrophilic than Gin), each of which are neutral or have a negative charge, and have a greater hydrophilicity value as compared to Gin Likewise, the statement "substitute Pro with a more hydrophilic ammo acid which is neutral or has a negative charge" means Pro would be substituted with Gin, Asn, Ser, Glu or Asp

In one embodiment of the present invention, the Thermitase variant has a modified ammo acid sequence of Thermitase wild-type am o acid sequence, wherein the wild-type ammo acid sequence comprises a substitution at one or more positions in one or more of the first loop region, the second loop region, the third loop region, the fourth loop region, the fifth loop region or the sixth loop region, whereby the Thermitase variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Thermitase

In a preferred embodiment of the present invention, the substituting am o acid for one or more of the positions in one or more of the loop regions is with reference to Table 1 , neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the ammo acid at the subject position in the wild-type ammo acid sequence A Substitutions in the First Loop Region

When a substitution occurs in the first loop region, the substitution occurs at one or more of positions 66, 67, 68, 69, 70, 72 or 73

When a substitution occurs at position 66, the substituting ammo acid is Asn Asp, Glu or Ser

When a substitution occurs at position 67, the substituting ammo acid is Asp, Gin, Glu or Ser

When a substitution occurs at position 68, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser.

When a substitution occurs at position 69, the substituting ammo acid

When a substitution occurs at position 70, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 72, the substituting ammo acid is Asn Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 73, the substituting am o acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser B Substitutions in the Second Loop Region

When a substitution occurs in the second loop region, the substitution occurs at one or more of positions 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114 or 115

When a substitution occurs at position 103, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr.

When a substitution occurs at position 104, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Met, Pro, Ser , Thr or Val.

When a substitution occurs at position 105, the substituting am o

When a substitution occurs at position 106, the substituting ammo acid is Asp, Gin, Glu or Ser

When a substitution occurs at position 107, the substituting ammo

When a substitution occurs at position 108, the substituting ammo acid is Asn, Asp Gin, Glu, Pro or Ser

When a substitution occurs at position 109, the substituting ammo

When a substitution occurs at position 110, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 111 , the substituting am o acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 112, the substituting ammo acid is Ala Asn Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Met, Phe, Pro, Ser, Thr Tyr or Val

When a substitution occurs at position 113, the substituting am o acid is Asn Asp, Gin Glu, Gly, Pro or Ser

When a substitution occurs at position 114, the substituting am o acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr

When a substitution occurs at position 115, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr C Substitutions in the Third Loop Region

When a substitution occurs in the third loop region, the substitution occurs at one or more of positions 134, 135, 136, 137, 138, 139, 140 or 141 wherein

When a substitution occurs at position 134, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Met, Pro, Ser, Thr or Val

When a substitution occurs at position 135, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 136, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 137, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 138, the substituting am o acid is Ala, Asn, Asp, cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr

When a substitution occurs at position 139, the substituting am o acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 140, the substituting ammo acid is Asp, gin, Glu or Ser and

When a substitution occurs at position 141 , the substituting ammo acid is Asp or Glu

D Substitutions in the Fourth Loop Region

When a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 162, 163, 164, 165, 166, 167, 168, 169, 170 or 171

When a substitution occurs at position 162, the substituting am o acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 163, the substituting am o acid is Asp, Gin, Glu or Ser

When a substitution occurs at position 164, the substituting am o acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr

When a substitution occurs at position 165, the substituting ammo acid is Asn Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 166, the substituting ammo acid is Asp, Gin, Glu or Ser

When a substitution occurs at position 167, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 168, the substituting am o acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr

When a substitution occurs at position 169, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly or Ser

When a substitution occurs at position 170, the substituting ammo acid is Asp, Gin, Glu or Ser and

When a substitution occurs at position 171 , the substituting am o acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val E Substitutions in the Fifth Loop Region

When a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 191 , 192, 193, 194 cr 195

When a substitution occurs at position 191 , the substituting ammo

When a substitution occurs at position 192, the substituting ammo

When a substitution occurs at position 193, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr, Tyr or Val

When a substitution occurs at position 194, the substituting ammo acid is Asp or Glu

When a substitution occurs at position 195, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser F Substitutions in the Sixth Loop Region

When a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 204, 205, 206, 207, 208, 209 210, 211 , 212 213, 214, 215, 216, 217, 218 or 219, 220, 221 , 223 or 224

When a substitution occurs at position 204, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr

When a substitution occurs at position 205, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly or Ser

When a substitution occurs at position 206, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 207, the substituting ammo

When a substitution occurs at position 208, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, Tyr or Val

When a substitution occurs at position 209, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val

When a substitution occurs at position 210, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val

When a substitution occurs at position 211 , the substituting ammo

When a substitution occurs at position 212, the substituting am o acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 213, the substituting ammo acid is Ala, Asn, Asp, Cys Gin, Glu, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr or Va!

When a substitution occurs at position 214, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, or Ser

When a substitution occurs at position 215, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser.

When a substitution occurs at position 216, the substituting ammo

When a substitution occurs at position 217, the substituting am o acid is Asn Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 218 the substituting ammo acid is Ala Asn Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val

When a substitution occurs at position 219, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr

When a substitution occurs at position 220, the substituting ammo

When a substitution occurs at position 221 , the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser, Thr or Val

When a substitution occurs at position 222, the substituting ammo acid is Asp or Glu

When a substitution occurs at position 223, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 224, the substituting am o acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser G Preparation of enzyme variants

Example 1 Mutant Thermitase DNA

A phagemid ("TP") containing the wild type Thermitase gene is constructed The 2 8 Kbp Pvu II restriction enzyme fragment of plasmid pUC119, (Vieira, J and Messing, J , "Production of Single-Stranded Plasmid DNA", 153 METHODS IN ENZYMOLOGY 3-11 (1989)) is cloned into the Pvu II site of plasmid pUB110 (Bacillus Genetic Stock Center, Columbus, OH 1 E9) The pUC119-pUB110 hybrid plasmid is named pJMA601 Into the 6amH1 restriction site of PJMA601 is cloned the polymerase chain reaction-amplified Thermitase gene gene giving TP Phagemid TP is transformed into Eschenchia coli Ung- strain CJ236 and a single stranded uracil-contaming DNA template is produced using the VCSM13 helper phage (Kunkel, T A , J D Roberts and R A Zakour, "Rapid and efficient site-specific mutagenesis without phenotypic selection", METHODS IN ENZYMOLOGY, Vol 154, pp 367-382, (1987), as modified by Yuckenberg, P D , F Witney, J Geisselsoder and J McClary, "Site-directed in vitro mutagenesis using uracil-contaming DNA and phagemid vectors", DIRECTED MUTAGENESIS - A PRACTICAL APPROACH, ed M J McPherson, pp 27-48, (1991 ), both of which are incorporated herein by reference) A single primer site-directed mutagenesis modification of the method of Zolier and Smith (Zoller M J and M Smith, "Ohgonucleotide-directed mutagenesis using M13-derιved vectors an efficient and general procedure for the production of point mutations in any fragment of DNA", NUCLEIC ACIDS RESEARCH, Vol 10, pp 6487-6500, (1982), incorporated herein by reference) is used to produce all mutants (basically as presented by Yuckenberg, et al , 1991 , above) Oligonucleotides are made using an Applied Biosystem Iπc 380B DNA synthesizer Mutagenesis reaction products are transformed into Escheπchia coli strain MM294 (American Type Culture Collection E coli 33625) All mutants are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain BG2036 (Yang, M Y , E Ferrari and D J Henner, (1984), "Cloning of the Neutral Protease Gene of Bacillus subtilis and the Use of the Cloned Gene to Create an In Vitro-deπved Deletion Mutation", JOURNAL OF BACTERIOLOGY, Vol 160, pp 15-21 ) For some of the mutants a modified TP with a frameshift-stop codon mutation in the corresponding loop is used to produce the uracil template Oligonucleotides are designed to restore the proper reading frame and to encode for random substitutions at positions 66, 67, 68, 69, 70, 72, 73, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 134, 135, 136, 137, 138, 139, 140, 141 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 191 , 192,193,194, 195, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223 or 224 (equimolar and/or variable mixtures of all four nucleotides for all three bases at these codons) Mutations that correct for the frameshift-stop and produce a functional enzyme are identified by their ability to digest casein The random substitutions are determined by DNA sequencing

Example 2 Fermentation The Bacillus subtilis cells (BG2036) containing a subtilism mutant of interest are grown to mid-log phase in a one liter culture of LB-glucose broth and inoculated into a Biostat ED fermenter (B Braun Biotech, Inc , Allentown, Pennsylvania) in a total volume of 10 liters The fermentation media contains Yeast Extract, starch, antifoam, buffers and trace minerals (see FERMENTATION A PRACTICAL APPROACH, Ed. B McNeil and L M Harvey, 1990) The broth is kept at a constant pH of 7 0 during the fermentation run Chloramphenical is added for antibiotic selection of mutagenized plasmid The cells are grown overnight at 37°C to an AβOO ° about 60 and harvested Example 3 Purification

The fermentation broth is taken through the following steps to obtain pure enzyme The broth is cleared of Bacillus subtilis cells by centrifugation, and clarified by removing fine particulates with a 100K cutoff membrane This is followed by concentration on a 10K cutoff membrane, and flow dialysis to reduce the ionic strength and adjust the pH to 5 5 using 0 025M MES buffer (2-(Λ/-morpholιno)ethanesulfonιc acid) The enzyme is further purified by loading it onto either a cation exchange chromatography column or an affinity adsorption chromatography column and eluting it from the column with a NaCl or a propylene glycol gradient (see Scopes, R K , PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, Spnnger-Verlag, New York (1984) incorporated herein by reference)

The pNA assay (DelMar, E G , C Largman, J W Brodrick and M C Geokas, ANAL BIOCHEM , Vol 99, pp 316-320, (1979), incorporated herein by reference) is used to determine the active enzyme concentration for fractions collected during gradient elution This assay measures the rate at which p-nitroanilme is released as the enzyme hydroiyzes the soluble synthetic substrate, succinyi-alanine-alanine-proline-phenylalanme-p- nitroanihde (sAAPF-pNA) The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration In addition, absorbance measurements at 280 nm are used to determine the total protein concentration The active enzyme/total-protein ratio gives the enzyme purity, and is used to identify fractions to be pooled for the stock solution

To avoid autolysis of the enzyme during storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column Upon completion of the purification procedure the purity of the stock enzyme solution is checked with SDS-PAGE (sodium dodecyl sulfate poiyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined via an active site titration method using trypsin inhibitor type ll-T turkey egg white purchased from Sigma Chemical Company (St Louis, Missouri) The measured conversion factors will show which changes made in the enzyme molecule at the various positions result in the enzyme variant having increased activity over the wild-type, against the soluble substrate pNA

In preparation for use, the enzyme stock solution is eluted through a Sephadex-G25 (Pharmacia, Piscataway, New Jersey) size exclusion column to remove the propylene glycol and exchange the buffer The MES buffer in the enzyme stock solution is exchanged for 0 1 M Tπs buffer (Tπs(hydroxymethyl-amιnomethane) containing 0 01 M CaCl2 and pH adjusted to 8 6 with HCl All experiments are carried out at pH 8 6 in Tπs buffer thermostated at 25°C H Characterization of enzyme variants

Example 4 Model Surface Preparation

Aminopropyl controlled pore glass (CPG) purchased from CPG Inc (Fairfield New Jersey) is used as a support for covalently attaching the sAAPF-pNA suDstrate purchased from Bachem, Inc (Torrence, California) The reaction is carried out in dimethyl sulfoxide and (1 -ethyl-3-[3- (dιmethylamιno)propylj carbodnmide hydrochloπde) (EDC) is used as a coupling agent Upon completion (monitored by pNA assay), the excess solvent is removed, and the CPG sAAPF-pNA is rinsed with dimethyl sulfoxide (DMSO) and doubly-distilled water This is followed by oven drying with a N2 purge at about 70°C The reaction scheme and preparation of the immobilized substrate are conducted as described by Brode, P F III, and D S Rauch, "Subtilism BPN' Activity on an Immobilized Substrate," LANGMUIR, Vol 8, p 1325-1329, (1992), incorporated herein by reference

The CPG surface will have 62,000 ± 7,000 pNA molecules/μm² The surface area will remain unchanged from the value of 50 0m²/g reported by CPG Inc for the CPG as received This suggests that the procedure used to add sAAPF-pNA to CPG does not damage the porous structure (mean diameter is 486 A)

Example 5 Surface Hydrolysis Assay

Using CPG sAAPF-pNA, adsorption of an enzyme variant and hydrolysis of a CPG-bound peptide can be measured in a single experiment A small volume of enzyme variant stock solution is added to a flask containing Tπs buffer and CPG sAAPF-pNA which has been degassed The flask is shaken on a wπst-action shaker for a period of 90 minutes during which the shaker is stopped at various time intervals (for example, every 2 minutes during the early stages of adsorption hydrolysis - e g , the first 20 minutes - and every 10 minutes towards the end of the experiment) The CPG sAAPF-pNA is allowed to settle and the solution is sampled Both the experimental procedure and the calculation of the adsorption and hydrolysis are conducted as described by Brode et al 1992 above

All enzymes are monitored for stability against autolysis and should show no appreciable autolytic loss over the time course of this experiment Therefore enzyme adsorption can be determined by measuring solution depletion The difference between the initial enzyme variant concentration and the concentration measured at each individual time point gives the amount of enzyme variant adsorbed The amount of pNA hydrolyzed from the surface is measured by taking an absorbance reading on an aliquot of the sample at 410 nm The total amount of pNA hydrolyzed is calculated by adding the amount sampled and the amount remaining in the flask This value is corrected by subtracting the amount of pNA that is hydrolyzed by Tπs buffer at pH 8 6 when no enzyme is present This base-hydrolysis ranges from 7-29% of the total hydrolysis depending on the efficiency of the enzyme

Example 6 Soluble Substrate Kinetic Analysis

The rates of hydrolysis of the soluble substrate sAAPF-pNA are monitored by measuring the adsorbance increase as a function of time at 410 nm on a DU-70 spectrophotometer The enzyme concentration is held constant and is prepared to be in the range of 6-10 nanomolar while the substrate concentration is varied from 90-700 μM sAAPF-pNA for each kinetic determination An adsorbance data point is taken each second over a period of 900 seconds and the data are transferred to a LOTUS™ spreadsheet (Lotus Development Corporation, Cambridge, Massachusetts) Analysis for kinetic parameters is conducted by the standard Lineweaver Burk analysis in which the data in the initial part of the run (generally the first minute) are fit to a linear regression curve to give v₀ The v₀ and s₀ data are plotted in the standard inverse fashion to give K^ and k_cat 58

15

I Example Thermitase variants

Thermitase variants of the present invention which have decreased adsorption to and increased hydrolysis of surface bound substrates are exemplified in Tables 2-36, below In describing the specific mutations, the original am o acid occurring in wild-type is given first, the position number second and the substituted ammo acid third TABLE 2 Loop 1 - Single Mutation Variants

GlnββAsn Gln66Asp GlnββGlu GlnββSer Asn67Asp Asn67Gln Asn67Glu Asn67Ser GlyβδAsn GlyβδAsp Gly68Gln Gly68Glu Gly68Pro Gly68Ser Asn69Asp Asn69Gln Asn69Glu Asn69Ser Gly70Asn Gly70Asp Gly70Gln Gly70Glu Gly70Pro Gly70Ser Gly72Asn Gly72Asp Gly72Gln Gly72Glu Gly72Pro Gly72Ser Thr73Asn Thr73Asp Thr73Gln Thr73Glu Thr73Gly Thr73Pro Thr73Ser 16

TABLE 3

Loop 1 - Double Mutation Variants

TABLE 4

Loop 1 - Triple Mutation Variants

G '.' ^" P e Thr73Ser Asr.6βSer Gly72Gln AsπόόSe T r73Gln Thr73Gin

Givc9Prc Thr73Ser Thr73Asn

A.sn£6Ser Thr73Asn Gl 69Asp Thr73Ser Asr.β8Asp Thr73Ser Asr.όβGln Thr73Asn Asr.58Ser Ser70Asp Asnβ8Gln Thr73Gln Glyc7Ser Gly72Glu Gly67Asn Thr73Asn Gl 7Gln Thr73Gln Glyc^Pro Gly69Pro Glyβ9Asn Thr73Ser AsnββSer Gly72Asn Glyβ9Gln Gly72Asn Gly€7Ser Thr73Gln AsnββGlu Thr73Pro Gly67Ser Thr73Ser Glyβ7Pro Gly72Asp AsnβoSer Thr73Pro AsnββSer AsnβδSer Gly67Asn Gly69Asp AsnβδGlu Gly72Pro AsnβδGlu Thr73Asn Gly67Glu Gly69Gln Gly57Glu Thr73Gln AsnββAsp Gly72Ser AsnββGlu

Thr73Pro

TABLE 5

Loop 1 - Quadruple Mutation Variants

Glyό7Asr. + AsnβδAsp + Gly69Asp + Ser70Glu

Gly67Asp + AsnβδGln + Gly69Glu + Ser70Asp

AsnββGin + Gly67Asp + Gly69Asp + Ser70Asp

Gly67A_sp + AsnβδAsp + Ser70Asp + Thr73Asn

Gly67Asp + AsnβδAsp + Gly69Pro + Ser70Glu

Gly67Asp + AsnβδGln + Ser70Glu + Gly72Pro

AsnββGin + Gly67Glu + Ser70Asp + Gly72Gln

Gly67Glu + Ser70Glu + Gly72Gln + Thr73Ser

Gly67Asp + AsnβδGln + Ser70Asp -r Gly72Ser

Gly67Glu + Ser70Asp + Gly72Ser + Thr73Ser

TABLE6

Loop 2 - Single Mutation Variants

Vall03Ala Vall03Asn Vall03Asp Vall03Cys Vall03Gln Vall03Glu Vall03Gly Vall03His Vall03Met Vall03Pro Vall03Ser Vall03Thr Leul04Ala Leul04Asn Leul04Asp Leul04Cys Leul04Gln Leul04Glu Leul04Gly Leul04His Leul04Ile Leul04Met Leul04Pro Leul04Ser Leul04Thr Leul04Val Aspl05Glu AsnlOβAsp AsnlOβGln AsnlOβGlu AsnlOβSer Serl07Asp Serl07Glu GlylOδAsn GlylOδAsp GlylOδGln GlylOδGlu GlylOδPro GlylOδSer Serl09Asp Serl09Glu GlyllOAsn GlyllOAsp GlyllOGln GlyllOGlu GlyllOPro GlyllOSer ThrlllAsn ThrlllAsp ThrlllGln ThrlllGlu ThrlllGly ThrlllPro ThrlllSer Trpll2Ala Trpll2Asn Trpll2Asp Trpll2Cys Trpll2Gln Trpll2Glu Trpll2Gly Trpll2His Trpll2Ile Trpll2Leu Trpll2Met Trpll2Phe Trpll2Pro Trpll2Ser Trpll2Thr Trpll2Tyr ϊrpll2Val Thrll3Asn Thrll3Asp Thrll3Gln Thrll3Glu Thrll3Gly Thrll3Pro Thrll3Ser Alall4Asn Alall4Asp Alall4Gln Alall4Glu Alall4Gly Alall4His Alall4Pro Alall4Ser Alall4Thr Valll5Ala Valll5Asn Valll5Asp Valll5Cys 20

Vail 15Gln Vail15Glu Vail15Gly Vail15His Vail15Met Vail15Pro Vail15Ser Vail15Thr

TABLE7

Loop 2 - Double Mutation Variants

21

G yl OoGln + Aial 14Gly Leul04Met T Vail15Glu Leu:04Gly + Alal14Ser Thr:13Ser + Vail15Ala

GlyllOGlu + Vail15Met Vail03Gly + GlyllOGlu Ser:09Asp + GlyllOGln Thr: ::ASD + Alal14Ser Asn: OβGlu + Alal14Pro Serl09Glu + Thrl13Gly GlylOδAsn + Trpl12Glu Vail03Pro + AsnlO^βGlu Aspl05Glu + Trpl12Leu Leul04Cys + Aspl05Glu Leu:04Cys + Thr:HSer Leu:04Thr + Ser:09Asp Vail03Asn + Vail15Glu Vail03Ser + GlylOδGlu

TABLE8

Loop 2 - Triple Mutation Variants

Va l03Gln - Leul04Thr + Serlo GΪTT

Leul04Ser + As_P:05Glu ₊ Trpll2Phe

AsnlO^βGln + GlylOδSer + GlyllOGln

Serl09Asp ₊ ThrlllAsn ₊ Trpli2Thr

⁺+ V^Sea?l^:l⁰l⁹5^AG^sl^pv

AsnlOGSer , Serl07Asp + ValllSGly

AsnlO^βSer + Trpll2Gln + ValllSGlu

Leul04Pro + Serl09Asp ₊ Thrll3Gly

Leul04Asp ₊ GlyllOGln ₊ ValllSPro

GlylOδAsn + ThrlllSer + Alall4Pro + ThrlllGlu

⁺ AsnlO^βAsp

AsnlO^βGln + Trpll2Asn + Alall4A n

Vall03Me ₊ GlyllOPro ₊ ThrlllPro

Leul04Thr ₊ GlylOδGlu ₊ GlyllOAsn

Leu:04Ala ₊ Serl09Asp + TrpT^Cvs

GlylOδGlu + GlyllOPro + ValllSGlv

Leul04Pro + Thrll3Asn + Alall4Asp

Vall03Asn + Leul04Met + AsnlO^βGlu

I' _n° ^^{s + s}erl09Glu ₊ ValllSCys

Vall03Gly + Leul04Val + Serl07Asp

GlylOδPro + Serl09Glu + Trpll2Gln

Leul04Met ₊ Trpll2Cys ₊ T A

AsnlOδAsp ₊ Trpll2lle ₊ Alall4Ser

Vall03Gly + Serl09Glu + Alall4Pro

Leul04Ile ₊ Trpll2Leu ₊ Thr:i3Gly

Asn:06Gln + GlylOδAsn + Serl09Glu ail03Asn ~ Trpl12Pro + Alal14Asp GlvlOδAsn + Glyl:0Ser + Trpl12Gly GlyllOAsp + Trpl12Leu + Vail15Hιs Vail03Gly + Leul04Gly + Glyl06Asp Leul04Ala + ThrlllSer + Vail15Ala HSer + Thrl13As + Vail15Hιs

A.splJ5G1U + GlylOδGln + Vail15Gly Leul04Gly 1- Alal14Ser -*- Vail15Ala Vail03Hιs - Leul04Gly + AsnlOβGlu Vail03Ser + GlyllOGlu -*- Thrl13Pro Vail03Pro + Glyl10Asn + Alal14Glu Leul04Met + GlyllOAsp + Vail15Pro Leul04Asp + ThrlHGln + Trpl12Ala Serl09Asp + GlyllOPro + Trpl 12Asn Leu:04Asp -*- Aspl05Glu + Trpl12Cys GlylOSGlu + Serl09Glu + Alal14Thr GlylOδAsp + Serl09Asp + ThrlHPro Glyl08Glu -*^■ Serl09Glu + Alal14Asn AsnlOβSer + Serl09Glu + GlyllOAsp Asn:OβGln + Serl09Asp + GlyllOAsp Vail03Asp + Leu:04Asp + Glyl08Pro Leul04Thr + Aspl05Glu + AsnlOβGlu Aspl05Glu + AsnlOβAsp + Thrl 13Asn GlylOδGln + Thrl13Glu + Alal14Glu Leul04Ser + ThrlHGlu + Trpl12Glu AsnlOβAsp + SerlO^Asp + GlylOδAsp Aspl05Glu + Serl09Asp + GlyllOGlu Trpl12Ala + Thrl13Asp + Vail15Asp Vail03Gln + Aspl05Glu + GlylOδAsp Leul04Gln + Aspl05Glu + Serl07Glu Serl07Asp + Serl09Asp + ThrlHGly Vail03Cys + Serl07Asp + Serl09Glu

TABLE 9

Loop 2 - Quadruple Mutation Variants

Aspl05Glu T GlyllOSer + Trpll2Phe + Thrll3Ser

Vall03Ala -^«- Serl07Asp + GlyllOPro + Alall4Asn

Vall03Gln + GlylOδPro + ThrlllSer + Alall4Glu

Vall03Thr + Serl07Asp + Thrll3Gln + Valll5Cys

Leul04Asn -*- GlyllOSer + Thrll3Glu + Valll5Gln

Vall03Pro -r GlylOδGlu + Trpll2Hιs + Valll5Met

Vall03Ala + AsnlOβSer + GlylOδAsp + ThrlllAsn

Vall03Ala + Leul04Asp + GlyllOSer + ValllδAla

Serl09Glu -*- Trpll2Pro + Alall4Asn + Valll5Met

Vall03Thr + AsnlOβAsp + GlylOδSer + GlyllOGln

Vall03Pro + Leul04Pro + AsnlOβSer + GlyllOAsn

Serl09Glu i- GlyllOPro + ThrlllSer + Thrll3Ser

AsnlOβGln -^»- GlylOδSer ^J- Trpll2Tyr + Alall4Thr

Leul04Cys + GlyllOGln + ThrlllAsp + Thrll3Gly

Leul04Ser + Aspl05Glu + GlylOδGln + Valll5Asn

Vall03Asp + GlylOδAsn + Thrll3Pro + Alall4Pro

Leul34Gln Leul34Glu Leul34Gly Leul34His Leul34Ile Leul34Met Leul34Pro Leul34Ser Leul34Thr Leul34Val Glyl35Asn Glyl35Asp Glyl35Gln Glyl35Glu Glyl35Pro Glyl35Ser Glyl36Asn Glyl36Asp Glyl36Gln Glyl36Glu Glyl36Pro Glyl36Ser Thrl37Asn Thrl37Asp Thrl37Gln Thrl37Glu Thrl37Gly Thrl37Pro Thrl37Ser Vall3δAla Vall36Asn Vall36Asp Vall3δCys Vall36Gln Vall36Glu Vall3δGly Vall3δHis Vall3δMet Vall38Pro Vall3δSer Vall3δThr Glyl39Asn Glyl39Asp Glyl39Gln Glyl39Glu Glyl39Pro Glyl39Ser Asnl40Asp Asnl40Gln Asnl40Glu Asnl40Ser Serl41Asp Serl41Glu TABLE 11

Loop 3 - Double Mutation Variants

26

Leul34Val 4 Thrl37Asp Glyl39Gln Leul34Hιs 4 Glyl35Pro Glyl39Gln Thrl3^Pro -*- Vail3δGln Asnl40Asp Thrl37Gln 4 Vail3δCys Glyl39Glu Thrl37Ser 4 Glyl39Glu Asnl40Gln Glyl35Gln -r Thrl37Gln Asnl40Asp Glyl35Gln 4 Thrl37Gln Vail 36Asp Leul34Hιs 4 Glyl39Gln Asnl 40Asp Leul34Ala 4 Thrl37Asn Glyl39Glu Vall38Pro 4 Glyl39Pro Asnl40Gln Glyl35Asn 4 A.snl40Ser Serl41Glυ Leul34Ile 4 Glyl35Asp Vail36Cys Thrl37Gln 4 Glyl39Ser Asnl40Ser Leul34Ala 4 Glyl36Pro Asnl40Asp Glyl35Ser -*- Glyl39Asp Asnl40Ser Leul34Thr 4 Glyl35Asn Serl41Glu Leul34Gly 4 Thrl37Ser Vail 38Ala Leul34Thr + Glyl35Gln Vail38Pro Glyl35Gln 4 Glyl36Asp Thrl37Ser Glyl35Ser 4 Vail3δThr Asnl 40Asp Leul34Cys 4 Thrl37Ser Vail38Glu Glyl36Ser 4 Thrl37Asn Serl 1Asp Leul34Thr 4 Glyl35Glu Glyl39Gln Leul34Thr Glyl35Asp Thrl37Ser

TABLE 13

Loop 3 - Quadruple Mutation Variants

Asnl40Ser Serl41Glu Thrl37Ser 4 Vall36Gln Vall38Ser 4 Glyl39Ser Thrl37Pro Asnl40Gln Glyl39Gln 4 Serl41Glu Glyl39Ser 4 Serl41Asp Thrl37Glu 4 Asnl40Gln Vall3δGln 4 Glyl39Ser Thrl37Asn Asnl40Ser Glyl36Ser 4 Thrl37Ser Glyl39Gln 4 Asnl40Glu Vall3δAsn 4 Asnl40Gln Thrl37Asn Serl41Glu Asnl40Gln 4 Serl41Asp Glyl39Asn Serl41Glu Vall3δThr Asnl40Asp Thrl37Ser Vall3δSer Asnl40Ser + Serl41Glu Vall38Cys 4 Glyl39Pro Thrl37Gln 4 Glyl39Glu Vall36Cys Serl41Glu Vall3δGln Serl41Asp Thrl37Asn 4 Asnl40Ser

Asnl40Gln 4 Serl41Asp Leu-34Met Glyl36Pro 4 Vail38Met Serl41Glu Leu 134Cys Thrl37Gln - Vail36Pro 4 Serl41Asp r i , , . u - v _36Asn Thrl37Gln 4 Glyl39Asn 4 Asnl40Ser 34Asn Thrl37Ser 4 Glyl39Ser 4 Asnl4OAsp 35Asp Glyl36Asp 4 Vail36Met Asnl40Ser

Glyl35Glu Glyl36Asp 4 Vail3δHιs Glyl39Gln Leul34Ser Glyl36Glu 4 Thrl37Glu Vail38Thr Leul34Thr Vall38Gln 4 Asnl40Glu Serl41Glu Glyl ;in Thrl37Asn 4 Asnl40Glu Serl41G1U Glyl -obin Thrl37Gly 4 Vail3δAsp 4 Glyl39Asp Leul34Thr Glyl35Glu 4 Glyl 36Asp Thrl37Asp Vail38Asn Glyl39Asp 4 Asnl40Glu Serl41Asp Leul34Ile Glyl39Giu 4 Asnl 4OAsp 4 Serl41Asp Glyl36Pro Glyl39Glu 4 Asnl40Glu Serl41Asp Leul34Gln Glyl39Asp 4 Asnl4OAsp 4 Serl41Glu Leul34Val Glyl36Glu 4 Vail3δAsp 4 Asnl40Gln Glyl36Asp Thrl37Gln 4 Vail3δAsp 4 Glyl39Asn Glyl36Glu Thrl37Glu 4 Vail3δHis 4 Glyl39 _sp Leul34Hιs Thrl37Gln 4 Vail3δGlu 4 Asnl40Glu Leu:34Gly Glyl35Ser 4 Thrl37Glu 4 Glyl39Glu Leu:34Gln Glyl35Pro 4 Thrl37Asp 4 Glyl39Asp Leu:34Hιs Glyl35Asn 4 Thrl37Asp 4 Glyl39Glu Leul34Val Glyl35Glu 4 Thrl37Asp 4 Glyl39Gln Glyl35Asn Thrl37Ser 4 Glyl39Asp Serl41Asp Leul34Cys Thrl37Asn 4 Glyl39Glu 4 Serl41Asp Leul34Ile Thrl37Pro 4 Glyl39Asp 4 Serl41Asp Leul34Thr Vall38Asn 4 Glyl39Glu 4 Serl41Asp Glyl35Pro Thrl37Gly 4 Glyl39Glu Serl41Asp Glyl36Ser Vall36Ala Glyl39Glu Serl41Glu Leul34Thr Glyl39Asp Asnl40Ser Serl41Asp Glyl35Ser Thrl37Gly Glyl39Glu Serl41Asp Leul34Met Glyl35Asn Glyl39Asp 4 Serl41Glu Thrl37Pro Vall36Gly Glyl39Asp Serl 1Asp Glyl36Glu Thrl37Pro Vail36Gln 4 Glyl39Asp Thrl37Asn Vall36Glu Glyl39Asp Serl41Asp Leul34Thr Vall36Glu Glyl39Glu Serl41Glu

TABLE 14

Loop 4 - Single Mutation Variants

Glyl 62Asn Glyl62Asp Glyl62Gln Glyl62Glu Glyl62Pro Glyl62Ser Asnl63Asp Asnl63Gln Asnl63Glu Asnl63Ser Alal64Asn Alal64Asp Alal64Gln Alal64Glu Alal64Gly Alal64His Alal64Pro Alal64Ser Alal64Thr Glyl65Asn GlylβSAsp Glyl65Gln Glyl65Glu Glyl65Pro Glyl65Ser AsnlββAsp AsnlββGln AsnlββGlu AsnlββSer Thrl67Asn Thrl67Asp Thrlβ7Gln Thrl67Glu Thrl67Giy Thrl67Pro Thrl67Ser AlalβδAsn AlalβδAsp AlalβδGln Alal68Glu AlalβδGly Alal68His AlalβδPro AlalβδSer AlalβδThr Prol69Asn Prol69Asp Prol69Gln Prol69Glu Prol69Gly Prol69Ser Asnl70Asp Asnl70Gln Asnl70Glu Asnl70Ser Tyrl71Ala Tyrl71Asn Tyrl71Asp Tyrl71Cys Tyrl71Gln Tyrl71Glu Tyrl71Gly Tyrl71His Tyrl71Ile Tyrl71Leu Tyrl7lMet Tyrl71Pro Tyrl7lSer Tyrl71Thr Tvrl71Val

TABLE 15

Loop 4 - Double Mutation Variants

Alal64Gln 4 Prol69Glu Asnl63Asp + Thrl67Ser Alal64Pro 4 AlalβδAsp Alal64Gln 4 AsnlββSer GlylβSAsp 4 Thrl67Pro Alal64Gln + Alal68Ser Glyl65Gln 4 AsnlββGln Glyl62Gln 4 Asnl63Glu Glyl65Gln 4 Thrl67Glu Glyl65Ser 4 AlalβδGln Alal64Gln 4 Alal68Glu Alal64His 4 Tyrl71Met Glyl65Pro 4 Prol69Gln Glyl65Gln 4 AlalβδPro Glyl62Pro 4 Alal64Gly Thrlβ7Pro 4 Asnl70Ser Asnl63Gln 4 Tyrl71His Asnl63Ser 4 Alal64Gln Alal64Asp AsnlββGln Glyl65Asn + Tyrl71Ile Alal68Asp 4 Asnl70Gln AsnlββAsp + AlalβδGly Asnl63Glu + Thrl67Ser AsnlββGlu 4 AlalβδSer Glyl62Gln 4 AlalβδGlu Glyl62Asn 4 Asnl70Ser Asnl63Gln + Alal68His Thrl67Pro 4 AlalβδThr Glylβ2Asn 4 Glyl65Glu AsnlββGlu Tyrl71Pro AlalβδAsn Asnl70Gln Glyl62Glu Thrl67Ser Thrlβ7Asp Tyrl71Val Glyl62Asp 4 AlalβδHis Glyl65Ser 4 AlalβδGlu Thrl67Asp 4 Tyrl71Met Alal68Thr 4 Prol69Gly Glyl65Ser Prol69Glu Glyl62Glu 4 Alal64Thr AlalβδSer Tyrl71Asp Asnl63Ser 4 Glyl65Pro AlalβδAsn Tyrl71Ser Alal64Gln 4 Glyl65Gln Thrl67Asp 70Ser GlyiβSSer 70Gln Thrl6"Gly ⁷1Glu Glyl62Asn 71Gln GlylβSAsn ββGlu AlalβδThr 71Glu Glyl65Ser ββGlu Thrl^β7Gly 68Thr AsnlββAsp 68Asn Prol69Glu 71Val Glyl62Glu 70Ser Glyl62Ser 69Gly Alal64Asn ββGlu Thrl67Gly 7OAsp Thrl67Glu 70Ser Asnl63Asp 64Gln GlylβδGln

69Glu

TABLE 16

Loop 4 - Triple Mutation Variants

Alal64Gly 4 GlylβSGlu 4 Thrl67Ser Glyl62Asn + Alal64Thr 4 AsnlββAsp Alal64Asn 4 Glyl65Asp 4 Prol69Gln Glyl65Pro 4 Thrl67Asn 4 Prol69Glu AlalβδGly 4 Prol69Gly 4 Asnl70Asp Glyl62Asp Glyl65Pro 4 AlalβδSer Glyl65Asp + Thrl67Asn 4 Tyrl71Gly Thrl67Ser Prol69Asn 4 Tyrl71Thr AsnlββSer 4 AlalβδGly 4 Tyrl71Ile Glyl65Gln 4 Thrl67Pro 4 AlalβδGlu Glyl62Ser 4 Asnl63Asp 4 Glyl65Gln GlylβSGlu 4 Thrl67Gly 4 AlalβδSer Thrl67Gly 4 AlalβδAsp 4 Tyrl71Val Asnl63Ser Glyl65Gln + AlalβδAsp Asnl63Asp + Alal64Gly Glyl65Asn Alal64Gln 4 Prol69Ser 4 Asnl70Gln Alal64His + Thrl67Gln 4 Prol69Gly Thrl67Ser AlalβδAsn 4 Tyrl71Asn Glyl62Asp 4 Glyl65Ser 4 AlalβδAsn Glylβ2Pro 4 Asnl63Gln 4 Tyrl71Val AsnlββGlu 4 AlalβδSer 4 Asnl70Gln Thrl67Pro Prol69Ser 4 Tyr:71His Glyl62Gln 4 Asnl63Gln 4 GlylβδAsp Alal64Ser Thrl67Ser 4 AlalβδGlu Thrl67Gln 4 Prol69Glu 4 Asnl70Gln Asnl63Gln Prol69Asn 4 Asnl70Gln Glyl62Glu AlalβδGln 4 Asnl70Gln Alal64Gly 4 Glyl65Ser 4 AsnlββAsp Alal64Ser 4 AlalβδGly Asnl70Gln Asnl63Glu + Alal64Asn 4 AlalβδSer Thrl67Ser 4 Prol69Glu Tyrl71Ser Alal βδGlu 4 Prol69Gln + Tyrl71Ile Glylδ2Pro 4 Alal64Pro Tyrl71Pro Glyl62Pro 4 AsnlββSer + Prol69Glu Alal64Gly 4 Alal 68Asn + Tyrl71Ser Alal β4Thr 4 Asnl 7OAsp Tyrl71Leu Asnl63Gln Thrl67Glu 4 Prol69Ser AsnloβAsp 4 Alal68Gln Tyrl71Pro Glyl52Asn 4 Alal64Glu 4 Tyrl71Ala AsnlββGlu 4 Thrl67Gln 4 Alal^βδPro A:a:64Pro 4 AlalβδGln 4 Asnl7OAsp Alal 64Thr 4 GlylβSGln 4 Alal6δAsp Alal64Ser AlalβδGly 4 Asnl70Glu Alal64His 4 Glyl65Glu 4 Tyrl71Met Alal64Gln Glyl65Asp + AsnlββGln Glylβ2Gln Thrl67Asn Alal 68Asn AlalβδHis 4 Prol69Gln 4 Tyrl71Glu Alal64Gln 4 AsnlββGln 4 Tyrl71Val Glyl62Pro 4 Alal64Ser 4 Asnl70Gln Thrl67Glu 4 AlalβδGly 4 Asnl70Ser Thrl67Ser 4 Prol69Gly 4 Asnl70Ser Asnl63Glu AsnlββSer 4 AlalβδSer Asnl63Gln 4 AlalβδGlu Tyrl71Ala Thrl67Ser 4 AlalβδSer 4 Asnl 7OAsp Glyl65Asp 4 AsnlββGlu 4 Tyrl71Pro Asnl63Asp 4 Alal64Asp Thrl67Ser Alal64 -sp 4 Glyl65Glu 4 AsnlββGln Glyl62Ser 4 Alal64Glu Glyl65Asp Glyl65Asn 4 Asnl70Glu 4 Tyrl71Glu Glvl65Pro 4 Asnl70Glu 4 Tyrl71Glu

TABLE 17

Loop 4 - Quadruple Mutation Variants

Alal64Asn AsnlββAsp 4 Thrlβ7Asn 4 AlalβδGln Glyl62Asn Asnl63Gln Alal64Asn 4 Asnl70Glu Alalβ4Thr 4 AsnlββSer 4 AlalβδAsn 4 Prol69Glu Alal64Gln 4 Prol69Asn 4 Asnl70Glu + Tyrl71Thr Asnl63Ser Alal64Thr 4 Thrlβ7Asp Tyrl71Thr Thrl67Ser Prol69Ser + Asnl70Asp 4 Tyrl71Ser Glyl62Asn 4 Thrl67Gly 4 Prol69Gln + Tyrl71Ser Glyl62Pro 4 Asnl63Ser 4 AlalβδGlu 4 Tyrl71Val Asnl63Gln 4 AlalβδAsp + Prol69Gln 4 Asnl70Gln Alal64Asn Glyl65Ser 4 Prol69Gln Tyrl71Ile Glyl62Glu 4 Thrl67Gln 4 AlalβδSer 4 Tyrl71Ser Glyl65Ser 4 AlalβδGly 4 Asnl70Asp 4 Tyrl71Leu Alalβ4His Glyl65Asp 4 AsnlββGln 4 Tyrl71Thr Asnl63Gln Alal64Ser AsnlββSer 4 AlalβδGlu AsnlββAsp 4 Thrl67Asn 4 Asnl70Ser 4 Tyrl71Thr Glyl62Asp Glyl65Gln 4 Asnl70Ser 4 Tyrl71Gln Glyl62Pro AlalβδAsn Prol69Glu 4 Tyrl71Gly AsnlββGln 4 Thrl67Gln 4 AlalβδThr 4 Asnl70Asp Asnl63Gln 4 Thrl67Asp 4 AlalβδSer Tvrl71Pro Alal64Pro 4 Thrl67Glu 4 Prol69Ser 4 Tyrl71Val Glyl65Asn + AsnlββGln 4 Prol69Gly + Asnl70Asp AsnlββSer 4 Thr:67Pro 4 Alal68Asn 4 Asnl70Glu Alal64Gly AlalβδAsn 4 Prol69Asp + Tyrl71Ala A.lal64Gln GlylβSGln 4 Thrl67Ser 4 Prol69Glu Asnl63Glu ^■>^■ Glyl65Ser 4 Thrl67Gln 4 AlalβδThr Glyl62Pro 4 Alal64Thr 4 Thrl67Ser 4 Prol69Gln Glyl62Gln Asnl63Glu 4 GlylβSGln 4 Thrl67Pro Asnl63Ser 4 Prol69Gly 4 Asnl70Glu 4 Tyrl71Ser Asnl63Gln 4 Glyl65Ser 4 Asnl70Asp 4 Tyrl71Leu Alal64His + Glyl65Pro 4 Prol69Ser 4 Asnl70Gln Glyl62Pro 4 AsnlββGln 4 AlalβδAsn 4 Asnl70Glu Asnl63Gln 4 Glyl65Pro 4 Thrl67Ser 4 Tyrl71Gly Asnl63Gln 4 Glyl65Asn 4 AsnlββSer 4 Tyrl71Cys Asnl63Asp 4 Glyl65Pro 4 AsnlββSer 4 Tyrl71Gly Alal64Pro ^■>^■ Thrlβ7Pro 4 Prol69Glu 4 Asnl70Ser Asnl63Asp 4 Alal64Glu 4 Prol69Gln 4 Tyrl71His Asnl63Glu + Alal64Glu 4 AlalβδGly + Tyrl71His Thrl67Asp 4 A.lalβδGlu + Prol69Gln 4 Asnl70Ser Alal64Asp 4 Glyl65Asp 4 AlalβδAsn 4 Prolβ9Gln Glyl62Ser 4 Alal64Gly 4 Asnl70Asp 4 Tyrl71Glu Asnl63Gln 4 Thrl67Asn 4 AlalβδAsp 4 Prol69Glu Asnl63Ser 4 Glyl65Pro 4 AlalβδAsp 4 Prol69Glu Glyl65Ser 4 AlalβδAsp 4 Prol69Glu 4 Asnl70Ser AsnlββGln 4 AlalβδGln 4 Prol69Asp 4 Asnl70Asp Alal64Gly 4 Thrl67Gly 4 Prol69Glu 4 Asnl70Asp Glyl62Pro 4 Alal64Gly 4 Prolβ9Asp 4 Asnl70Glu Alal64Pro 4 Thrl67Glu 4 AlalβδAsp 4 Prol69Asp Glyl62Asp 4 Asnl63Gln 4 AlalβδHis 4 Asnl70Asp Glyl62Glu 4 Alal64Ser 4 AsnlββSer 4 Asnl70Glu Glyl62Glu 4 Alal64Ser 4 AsnlββGln 4 Asnl70Glu Thrl67Asp 4 AlalβδHis 4 Prol69Glu 4 Tyrl71Pro Asnl63Glu 4 Alal64His 4 Glyl65Asp 4 AsnlββGln Asnl63Asp Alal64Gln 4 Glyl65Asp 4 AsnlββSer Asnl63Glu 4 Alal64Asp Glyl65Gln 4 AlalβδAsp Glyl62Glu 4 Alal64Glu + AsnlββSer 4 Prol69Asp Alal64Glu 4 AsnlββAsp 4 Asnl70Ser 4 Tyrl71Gly Alal64Glu 4 Glyl65Gln 4 AsnlββGlu 4 Tyrl71Cys Alal64Asp 4 AsnlββGlu 4 Thrl67Asn 4 Prolβ9Asn Thrl67Asp 4 Prol69Glu 4 Asnl70Glu 4 Tyrl71Ser AsnlββAsp 4 Thrl67Ser 4 AlalβδAsp 4 Tyrl71His

TABLE 18

Loop 5 - Single Mutation Variants

Serl91Asp Serl91Glu Serl92Asp Serl92Glu Phel93Ala Phel93Asn Phel93Asp Phel93Cys Phel93Gln Phel93Glu Phel93Gly Phel93His Phel93Ile Phel93Leu Phel93Met Phe:93Pro Phe:93Ser Phe:93Thr Phel93Tyr Phel93Val Serl94Asp Serl94Glu Thrl95Asn Thrl95Asp Thrl95Gln Thrl95Glu Thrl95Gly Thrl95Pro Thrl95Ser

TABLE 19

Loop 5 - Double Mutation Variants

3

TABLE 20

Loop 5 - Triple Mutation Variants

Serl91Glu ÷ Serl92Glu 4 Phel93Cys 4 Thrl95Pro Serl91Asp Ser:92Glu 4 Phel93Cys + Thrl95Gln Serl91Asp - Ser:92Glu 4 Phel93Thr + Thrl95Pro Ser:9:Glu -r Serl92Glu 4 Phel93Val 4 Thrl95Asn 3er:91A_sp - Serl92Glu 4 Phel93Val 4 Thrl95Asn 5erl91Glu -*^■ Serl92Glu 4 Phel93Leu + Thr:95Ser Ser:9:Glu -^*- Serl92Asp 4 Phel93Cys + Thrl95Gln Serl91Glu + Serl92Asp + Phel93Pro 4 Thrl95Pro Serl91Glu - Serl92Asp 4 Phel93Ser 4 Thrl95Gly Serl91Glu 4 Serl92Asp 4 Phel93Pro Thrl95Asn Serl91Glu 4 Serl92Glu 4 Phel93Ser 4 Thrl95Pro Serl91Glu 4 Serl92Asp 4 Phel93Ala 4 Thrl95Gly Serl91Asp Serl92Glu 4 Phel93His 4 Thrl95Asn Serl91Asp -r Serl92Glu 4 Phel93Asn 4 Thrl95Asn Serl91Glu + Serl92Glu -» Phel93Gln 4 Thrl95Ser Serl9:Glu 4 Serl92Glu 4 Phel93Thr + Thrl95Gln Serl91Giu 4 Serl92Glu 4 Phel93Asn + Thrl95Gln Serl91Glu -*- Serl92Glu 4 Phel93Tyr 4 Thrl95Ser Serl91Asp 4 Serl92Glu 4 Phel93Ile 4 Thrl95Pro Serl91Glu 4 Serl92Asp + Phel93Val 4 Thrl95Gln Serl91Glu 4 Serl92Asp 4 Phel93Val + Thrl95Asn Serl91Asp 4 Serl92Glu + Phel93Tyr 4 Thrl95Ser Serl91Asp + Serl92Glu 4 Phel93Ala + Thrl95Ser Serl91Glu 4 Serl92Glu 4 Phel93His + Thrl95Asn Serl91Asp 4 Serl92Asp 4 Phel93Pro 4 Thrl95Asn Serl91Asp 4 Ser:92Asp 4 Phe:93Gln + Thrl95Ser Serl91Asp 4 Serl92Asp 4 Phel93Leu 4 Thrl95Asn Serl91Asp 4 Serl92Glu 4 Phel93Val + Thrl95Gln Serl91Glu 4 Serl92Glu 4 Phel93Gly Thrl95Gly Serl91Glu 4 Serl92Glu 4 Phel93Thr Thrl95Pro Serl91Glu 4 Serl92Glu 4 Phel93Thr 4 Thrl95Gly Serl91Glu 4 Serl92Asp + Phel93Ile 4 Thrl95Ser Serl91Glu 4 Serl92Glu 4 Phel93Ile + Thrl95Gly Serl91Asp 4 Serl92Asp 4 Phel93Pro 4 Thrl95Pro Serl91Asp 4 Serl92Asp 4 Phel93Ile Thrl95Gly Serl91Glu 4 Serl92Asp 4 Phel93Thr 4 Thrl95Gly Serl91Asp 4 Serl92Glu 4 Phel93Ser 4 Thrl95Gly Serl91Glu 4 Serl92Asp 4 Phel93Tyr 4 Thrl95Ser Serl91Asp 4 Serl92Asp Phel93Thr 4 Thrl95Gln Serl91Asp 4 Serl92Glu 4 Phel93Ser 4 Thrl95Gln Serl91Glu 4 Serl92Glu + Phel93His + Thrl95Ser Serl91Glu 4 Serl92Asp 4 Phel93Leu 4 Thrl95Asn Serl91Asp 4 Serl92Glu 4 Phel93Leu + Thrl95Pro Serl91Asp 4 Serl92Asp 4 Phel93Ile + Thrl95Pro Serl91Asp 4 Serl92Asp 4 Phel93Tyr 4 Thrl95Ser Serl91Asp + Serl92Glu 4 Phel93Ile 4 Thrl95Gln Serl91Asp 4 Serl92Glu 4 Phel93Pro 4 Thrl95Ser Serl91Asp + Serl92Glu -r Phel93Leu 4 Thrl95Gln Serl91Glu 4 Serl92Asp 4 Phel93Asn 4 Thrl95Ser Serl91Glu 4 Serl92Glu + Phel93Ser 4 Thrl95Asn Serl91Asp 4 Serl92Asp 4 Phel93Glu 4 Thrl95Gln Serl91Asp 4 Serl92Glu 4 Phel93Glu 4 Thrl95Pro Serl91Glu 4 Serl92Asp 4 Phel93Glu 4 Thrl95Gln

TABLE 22

Loop 6 - Single Mutation Variants

Ala204Asn Ala204Asp Ala204Gln Ala204Glu Ala204Gly Ala204Hιs A_la204Pro Ala204Ser Ala204Thr Pro205Asn Pro205Asp Pro205Gln Pro205Glu Pro205Gly Pro205Ser Gly206Asn Gly206Asp Gly206Gln Gly206Glu Gly206Pro Gly20βSer Ser207Asp Ser207Glu Trp208Ala Trp20δAsn Trp20δAsp Trp208Cys Trp20δGln Trp206Glu Trp208Gly Trp20δHis Trp20δlle Trp208Leu Trp208Met Trp206Phe Trp20δPro Trp20δSer Trp208Thr Trp20δTyr Trp20δVal Ile209Ala Ile209Asn Ile209Asp lle209Cys Ile209Gln Ile209Glu Ile209Gly Ile209His Ile209Leu Ile209Met Ile209Pro Ile209Ser Ile209Thr Ile209Val Tyr210Ala Tyr210Asn Tyr210Asp Tyr210Cys Tyr210Gln Tyr210Glu Tyr210Gly Tyr210His Tyr210Ile Tyr210Leu Tyr210Met Tyr210Pro Tyr210Ser Tyr210Thr Tyr210Val Ser211Asp Ser211Glu Thr212Asn Thr212Asp Thr212Gln Thr212Glu Thr212Gly Thr212Pro Thr212Ser Tyr213Ala Tyr213Asn Tyr213Asp Tyr213Cys Tyr213Gln Tyr213Glu Tyr213Gly Tyr213His Tyr213Ile Tyr213Leu Tyr213Met Tyr213Pro Tyr213Ser Tyr213Thr Tyr213Val Pro214Asn Pro214Asp Pro214Gln Pro214Glu Pro214Gly Pro214Ser Thr215Asn Thr215Asp Thr215Gln Thr215Glu Thr215Gly Thr215Pro Thr215Ser Ser216Asp Ser216Glu Thr217Asn Thr217Asp Thr217Gln Thr217Glu Thr217Gly Thr217Pro Thr217Ser Tyr216Ala Tyr21δAsn Tyr216Asp Tyr21δCys Tyr21δGln Tyr216Glu Tyr21δGly Tyr21δHis Tyr218Ile Tyr218Leu Tyr21δMet Tyr216Pro Tyr21δSer Tyr21δThr Tyr218Val Ala219Asn Ala219Asp Ala219Gln Ala219Glu Ala219Gly Ala219His Ala219Pro Ala219Ser Ala219Thr Ser220Asp Ser220Glu Leu221Ala Leu221Asn Leu221Asp Leu221Cys Leu221Gln Leu221Glu Leu221Gly Leu221His

TABLE 23

Loop 6 - Double Mutation Variants

Ala204Gly 4 Thr215Glu 42

Thr212Gln -*- Ala219Glu Ala204Gly + Trp208Gln Ala20 Thr 1βA.sp Thr212C-ln -*- Tyr2:6Gln Ser207Glu 4 Leu221Met Thr215Pro 4 Gly223Gln Ala204Gly 4 Ser2lβAsp Tyr210Asp Ala219His Ile209Gln 4 Tyr2IδAla Ile209Ala 4 Ala219Pro Ala204Asn -*- Ser222Glu Tyr210Asp + Tyr213Ala Tyr21δLeu 4 Thr224Glu Tyr216Asn 4 Ser220Glu Trp206Met + Ala219Pro Pro205Gly 4 Ser222Asp Leu221Val 4 Thr224Gln Trp206Ile + Pro214Ser Trp2OδAsp 4 Thr212Pro Ala204Thr 4 Gly223Gln Thr217Gln 4 Tyr2lδlle Tyr213His 4 Ser2lβAsp Thr212Ser -*- Thr215Asp Thr217Asn 4 Leu221Asn Pro214Asp 4 Thr217Pro Ser207Glu 4 Ala219His Thr217Asp 4 Tyr2lδHis Trp208Ala 4 Thr217Ser Pro205Gln 4 Ser2 2OAsp Ile209Gly 4 Ser2lβGlu Tyr218Ser 4 Ser220Glu Thr215Asn 4 Ser2lβGlu Thr215Gly 4 Tyr2lδGln Thr217Ser 4 Leu221Ala Ala219Pro 4 Leu221His Pro205Gln 4 Leu221Met Tyr213Met 4 Leu221Ile Trp208Asn Thr212Ser Ser207Glu 4 Thr224Gly Pro205Ser 4 Leu22ICys Ala204Gly 4 Tyr210Val Trp20δPhe Ser222Asp Ile209Thr 4 Ser222Glu Leu221Ile 4 Ser222Glu Tyr213Pro -*- Thr217Glu Ile209Thr Ser2lβGlu Tyr210Cys 4 Ser222Asp Leu221Glu 4 Thr224Ser Tyr213Ala 4 Thr217Pro Trp206Cys Thr215Glu Ala204Ser 4 Thr217Ser Ile209His 4 Gly223Ser Ala204Asn 4 Trp2OδTyr

Gly206Pro + Ser222Glu Ala204Thr 4 Ser220Asp Ala219Thr 4 Thr224Pro Trp20811e 4 Ala219Glu Ile209Gly 4 Thr215Ser Gly20βGln 4 Tyr210Glu Pro214Gln + Ser220Glu Ser207Glu 4 Thr217Asn Ala204Pro 4 Gly223Asp Ile209Cys 4 Ser222Asp Pro214Gly 4 Gly223Pro Ile209Met 4 Thr224Pro Pro214Glu 4 Thr217Ser Pro205Ser 4 Ser207Asp Tyr210Thr 4 Thr212Gln Gly206Gln 4 Tyr21δPro Tyr218Val 4 Ser222Glu Leu221Asn 4 Gly223Glu Pro205Gln 4 Tyr218Val Pro205Gln + Thr217Asn Trp206His 4 Ile209Ala Pro205Ser 4 Gly223Gln Pro214Ser + Thr217Gly Tyr216His 4 Ser222Asp Thr212Gln 4 Gly223Ser Thr215Glu 4 Ala219Thr Tyr213Gly 4 Tyr21δAsp Thr212Gln 4 Tyr213Gln Tyr210Ile + Thr215Asp Ala204Asn 4 Gly223Pro Ser207Glu 4 Ile209Asn Thr215Gln 4 Ala219Asp Ser220Asp 4 Gly223Pro Pro205Ser + Tyr213Ala Trp20δGly 4 Ser222Glu Thr215Glu 4 Tyr216Ala Ala204Gln 4 Ala219Gln Pro205Asn 4 Tyr213Thr Tyr21δThr 4 Thr224Gln Ser222Asp 4 Thr224Ser Ala204Pro 4 Tyr213Cys Gly20βGln 4 Leu221Gly Ser216Glu 4 Thr224Ser Ser207Asp 4 Ala219Asn Trp20δLeu 4 Ile209His Pro205Ser 4 Tyr213Val Thr212Gly 4 Thr215Asp

TABLE24

Loop 6 - Triple Mutation Variants

Gly206Ser 4 Thr212Ser 4 Ala219Glu Ala204Asn - Thr212Ser 4 Ser216Glu

Pro2I.5Gln - Leu221Glu 4 Gly223Asn

Trp2'_SThr 4 ϊle209Cys + Thr224Asp lle209Met - Thr212Gln + Ser220Asp

Tyr210Gly -r Tyr213Gly -*- Thr224Gly

Ser2?7Glu -r Tyr210Ser 4 Gly223Asn

Tyr213Gly Ser216Asp + Thr217Ser

Pro205Asn -r Ser216Glu 4 Thr224Asn

Thr215Glu 4 Ala219Gln 4 Leu221Thr

Ala204Asn 4 Thr2:7Asn 4 Tyr2:δGlu

Thr212Ser 4 Thr217Gly 4 Thr224Gln

Ala204Ser 4 Tyr21δHιs 4 Ala219Gln

Pro205Ser 4 Tyr213Ser 4 Leu221Asn

Ala204Ser 4 Gly206Ser 4 Ala219Ser

Ala204Gly 4 Trp208Gln 4 Ser216Asp

Ser207Glu 4 Tyr218Gln 4 Leu221Met

Ala204Gly 4 Thr215Pro 4 Gly223Gln

Ile209Gln 4 Tyr216Ala 4 Ala219Pro

Ala204Asn 4 Trp208Met 4 Ser222Glu

Trp208Met 4 Ala219Pro 4 Ser222Asp

Pro205Gly 4 Leu221Val Thr224Gln

Ala204Thr 4 Thr212Pro 4 Gly223Gln

Gly206Gln 4 lle209Gln 4 Thr212Gln

Gly206Pro + Ser220Glu 4 Leu221Ser

Gly206Gln 4 Tyr210Gly 4 Ser222Asp

Ala204Gly 4 Tyr213Thr 4 Pro214Gln

Ala204His 4 Thr212Gln 4 Thr224Gln

Gly206Gln 4 Pro214Asp 4 Tyr21δSer

Thr212Asn 4 Ser216Asp 4 Thr217Ser

Ser207Asp 4 Tyr213Ile 4 Thr224Pro

Pro205Gln 4 Tyr213Met 4 Leu221His

Trp20δAsn 4 Thr212Ser 4 Gly223Asp

Ser207Glu 4 Leu221Gly + Thr224Gly

Pro205Ser 4 Tyr210VaI 4 Leu221Cys

Pro205Gly 4 Ile209His 4 Tyr213Thr

Ile209Pro 4 Tyr210Asn 4 Ser220Asp

Pro205Gly 4 Thr212Pro 4 Thr224Gly

Trp20δCys 4 Thr217Asp 4 Ala219Asn

Pro205Gly 4 Leu221Cys 4 Gly223Pro

Ala204His 4 Ser216Glu 4 Leu221Ser

Thr217Asp 4 Gly223Ser 4 Thr224Gly

Pro205Gln 4 Trp20δGln 4 Ile209Ala

Thr212Gly 4 Thr217Gly 4 Leu221His

Gly206Asn 4 Thr212Gly 4 Tyr213Gly

Gly206Asn 4 Ser207Glu 4 Thr212Ser

Thr212Gln 4 Leu221Glu 4 Gly223Ser

Tyr210His 4 Thr215Asp 4 Thr224Pro

Pro214Gln 4 Thr215Pro 4 Ser216Glu

Pro205Gly 4 Tyr213Val 4 Gly223Asp

Trp20δPhe + Thr215Asn 4 Thr224Gly

Tyr213Met 4 Ala219Pro 4 Ser220Glu

Trp208Phe 4 Ile209Ser 4 Tyr213Gln

Gly206Ser 4 Ser216Glu 4 Tyr218Pro Ala204Gln + Tyr210His 4 Thr224Glu Pro205Ser + Thr212Pro 4 Ala219Ser Pro205Gln 4 Tyr210Met 4 Thr217Pro Ala204Gly + Ile209His 4 Ser216Glu Thr212Asn + Thr217Ser 4 Thr224Asn Gly206Pro + Tyr210Gly 4 Gly223Gln Pro205Gln 4 Thr217Asp 4 Ala219Thr Pro205Ser 4 lle209Met 4 Ser216Asp Thr215A.sn + Ser216Glu 4 Thr224Ser Pro205Gln 4 Leu221Thr 4 Ser222Glu Tyr210Cys 4 Ala219Asn 4 Leu221Thr Ala204Ser 4 Gly206Ser 4 Ala219Gln Ile209Pro 4 Ser216Asp 4 Leu221Cys Trp20δThr 4 Tyr210Val + Thr212Ser Thr212Gly 4 Pro214Asp 4 Gly223Gln Pro214Gln 4 Thr215Ser + Thr224Glu Thr212Ser 4 Pro214Gln 4 Leu221His Trp206Gly 4 Ser220Glu 4 Gly223Ser Pro205Ser 4 Ser220Asp 4 Leu221Thr Gly206Asn 4 Thr215Pro 4 Leu221Ala Tyr213Leu 4 Leu221His 4 Gly223Asp Ser207Asp 4 Thr212Gly 4 Tyr21δCys Gly20βGln 4 Ile209Val 4 Ala219Thr Trp206Ile 4 Tyr218Ile 4 Gly223Asn Ala204His 4 Ile209Gly 4 Ser2lβAsp Trp20δLeu 4 Thr212Gly 4 Ala219Ser Thr212Gln 4 Pro214Glu 4 Ala219Thr Pro205Gln 4 Ser207Asp 4 Thr212Pro Pro205Ser 4 Tyr210Pro Tyr216Ile Leu221Ala + Gly223Asn 4 Thr224Glu Ala204Thr + Gly206Gln 4 Ser207Asp Ser216Glu 4 Ala219Gln 4 Gly223Gln Pro205Gly 4 Trp20δLeu 4 Leu221Gln Thr217Gln 4 Gly223Gln 4 Thr224Glu Thr212Gly + Thr217Ser 4 Tyr216Ala Gly206Asn 4 Gly223Asp 4 Thr224Asn Ala204Gly Tyr210Cys 4 Tyr213Thr Gly206Ser 4 Thr212Asn + Thr215Gly Ala204Asn 4 Thr212Asn 4 Ser216Glu Gly206Gln 4 Tyr21δCys 4 Gly223Glu Ala204Ser 4 Pro205Ser 4 Gly206Asn Ile209Gly + Leu221Val Thr224Pro Gly206A_sn 4 Trp20δHis Thr217Gly Gly206Ser 4 Trp206Leu 4 Ser222Asp Pro205Gly + Thr212Gln + Ser222Asp Ala204Thr 4 Tyr213Gln + Thr224Pro Thr212Ser Thr217Ser + Gly223Asp Pro205Ser 4 Tyr213Ile + Ser222Glu Ile209Pro 4 Tyr210Asp Ala219Pro Gly206Gln 4 Thr215Pro + Ser220Glu Trp20δCys + Tyr210Leu 4 Thr215Gly Trp206Tyr 4 Thr212Gln 4 Ser222Glu Gly206Asn 4 Ser222Asp 4 Gly223Asn Pro205Gly 4 Gly206Asn 4 Leu221Cys

Pro205Gln 4 Tyr213Ile 4 Ser216Glu

Ala204Hιs 4 Tyr210Hιs 4 Ser220Asp

Ala204Thr 4 Thr212Asn 4 Ala219Asp

Ser207Glu 4 Tyr210Ala 4 Thr212Gly

Tyr210Hιs 4 Thr217Gly 4 Leu221Met

Ala204Thr 4 Ile209Met 4 Ser222Glu

Trp208Pro 4 Ala219Pro 4 Ser222Asp

Ala204Hιs 4 Ile209Ser 4 Ser220Glu

Pro214Ser 4 Leu221Glu 4 Gly223Gln

Gly206Pro 4 Leu221Met + Gly223Asp

Ala204Hιs 4 Tyr213Thr + Ser222Asp

Tyr213Ala + Leu221Met + Ser222Asp

Trp208Leu 4 Thr212Ser + Pro214Asp

Tyr213Ile -r Ser216Glu + Thr217Pro

Pro205Gln 4 Pro214Gln 4 Thr224Gly

Tyr213Cys 4 Thr217Gly 4 Ser220Glu

Gly206Asn 4 Thr212Gly 4 Tyr21δPro

Gly206Pro 4 Ser216Glu 4 Gly223Pro

Thr212Gln 4 Thr215Gly T Ser222Glu

Ala204Pro 4 Thr217Asp Thr224Ser

Pro205Ser 4 Thr212Pro 4 Ala219Asp

Ile209Cys 4 Pro214Ser 4 Thr215Asp

Ile209Cys 4 Ser220Asp 4 Gly223Gln

Thr217Gly Ser220Asp 4 Leu221Gln

Ala204Hιs 4 Pro205Asn 4 Trp20δGlu

Ala204Gln 4 Thr212Ser 4 Thr215Asp

Pro214Asn 4 Ser220Glu 4 Leu221Gly

Ile209Gly 4 Thr215Asn 4 Ala219Pro

Ser207Glu 4 Thr217Gln 4 Thr224Ser

Ala204Gly 4 Thr215Asn 4 Gly223Pro

Ile209Ser 4 Tyr213Ile Leu221Met

Tyr210Thr 4 Pro214Ser Ser222Asp

Tyr210Leu 4 Thr212Ser 4 Leu221Asn

Gly206Asn 4 Pro214Asp 4 Tyr21δMet

Gly206Asn 4 Pro214Gln 4 Ala219Asn

Ser207Glu 4 Pro214Ser 4 Ala219His

Ala204Asn 4 Trp206Ala 4 Ile209Gln

Thr217Glu 4 Tyr21δLeu 4 Gly223Asn

Trp206Pro 4 Pro214Gly Leu221Ala

Gly20βPro 4 Thr215Ser 4 Leu221Cys

Ala204Ser 4 Ile209Val 4 Thr224Asp

Trp20δlle 4 Tyr210Pro 4 Thr224Asp

Thr212Gln 4 Thr215Asn 4 Thr217Asp

Tyr210Ser 4 Tyr213Cys 4 Thr217Asn

Thr212Gln 4 Ser222Asp 4 Gly223Gln

Tyr210Asn 4 Thr217Glu 4 Tyr21δMet

Pro214Asn 4 Ala219Gln 4 Ser220Glu

Pro205Ser 4 Trp20δMet 4 Leu221Asp

Ala204Asn 4 Tyr210Cys 4 Tyr213Asn

Tyr21δLeu 4 Leu221His 4 Ser222Glu

Ala204His 4 Tyr216Ser 4 Ser222Asp

Gly206Pro 4 Tyr213Asn 4 Pro214Asn Gly206Pro + Thr215Gln 4 Tyr21δSer Ala204Pro - Ile209His + Leu221Thr Gly2j6Gln - Thr212Gly + Gly223Glu Thr215Gly - Tyr21δAla 4 Thr224Pro Prc2j5Gln - Tyr213Gly + Gly223Glu A_la204Gly 4 Tyr213Asn + Leu221Thr Trp208Gly + l!e209Hιs 4 Thr215Ser Ala204His 4 Ser207Glu + Tyr213Cys Trp208His 4 Tyr210His 4 Ser222Asp Pro205Gly Tyr210Asp + Leu221Gln Pro205Asn 4 Tyr210Ala 4 Pro214Ser Thr212Pro 4 Thr215Gln 4 Thr224Asp Ala204Thr r Tyr213Val + Pro214Glu Ala204Gly -r Tyr210Pro 4 Pro214Glu Pro205Gly Trp206His + Tyr218Gly Gly20βSer 4 Ala219Gln 4 Gly223Ser Tyr213Thr 4 Tyr218Cys 4 Ser220Glu Thr215Pro 4 Ser216Glu 4 Leu221Asn Pro205Gly Thr212Asn 4 Thr224Asp Pro205Gly + Ser207Asp 4 Thr212Ser

TABLE25

Loop 6 - Quadruple Mutation Variants

Ala204Gln 4 Trp20δAla 4 Tyr210Asn 4 Ser222Glu Gly206Gln 4 Ser220Asp + Leu221Ala 4 Thr224Ser Ile209Gln 4 Thr212Ser + Ala219Gln 4 Ser220Glu Pro205Asn 4 Thr212Gly + Tyr218Ala Ser222Glu Pro205Gln + Trp208Cys + Tyr213Cys 4 Pro214Asp Pro205Gly + Thr215Gln + Thr217Pro 4 Gly223Ser Ala204His 4 Gly206Pro + Ser216Glu 4 Leu221Ser Ser207Asp 4 Leu221Gly 4 Gly223Gln 4 Thr224Asn Tyr210His + Thr212Ser 4 Leu221Glu 4 Gly223Ser Pro205Gly Thr215Asn 4 Gly223Asp 4 Thr224Gly Trp20δPhe 4 Tyr213Met 4 Thr215Pro 4 Ser220Glu Gly206Ser 4 Trp208Phe 4 Ile209Ser 4 Tyr213Gln Ala204Gln 4 Pro205Ser 4 Thr212Pro 4 Ala219Ser Ala204Gly 4 Tyr210Met 4 Ser216Glu 4 Thr217Pro Gly206Pro + Thr212Asn + Thr217Gln 4 Thr224Asn Pro205Ser 4 Ile209Met 4 Thr215Asn 4 Thr224Ser Tyr210Thr 4 Thr212Pro 4 Tyr218Asn 4 Ser220Glu Trp208Thr 4 Tyr210Val 4 Thr212Ser 4 Leu221Cys Thr212Gly 4 Pro214Asp 4 Thr215Ser 4 Gly223Gln Ser207Glu 4 Thr212Ser 4 Pro214Gln 4 Leu221Pro Pro205Asn 4 Gly206Gln 4 Ser207Asp 4 Leu221Thr Pro205Ser 4 Thr215Pro 4 Ser220Asp 4 Leu221Cys Gly206Asn 4 Tyr213Leu 4 Leu221Ala 4 Gly223Asp Ser207Asp 4 Thr212Gly 4 Pro214Asn 4 Thr224Asn Ala204Pro 4 Ser207Glu 4 Ile209Val 4 Ala219Thr Gly206Gln Trp20δlle 4 Tyr216Ile 4 Gly223Asn Ala204His 4 Trp206Leu 4 Ile209Gly 4 Ser216Asp Ala204Gln 4 Thr212Gly 4 Pro214Glu 4 Ala219Ser Ala204Asn 4 Pro205Gln 4 Ser207Asp 4 Thr212Pro Pro205Gly + Trp20δLeu 4 Ser21βGlu Ala219Gln Trp20δTyr 4 Thr217Gln 4 Leu221Gln 4 Thr224Glu Thr212Gly 4 Thr217Ser 4 Tyr218Ala Gly223Asp Ala204Gly 4 Gly206Asn 4 Thr215Asn 4 Gly223Asp Gly206Ser 4 Tyr210Cys 4 Tyr213Thr 4 Thr215Gly Ala204Gln 4 Pro205Ser 4 Gly206Asn 4 Ala219Asp Ala204Ser 4 Ile209Gly Leu221Val 4 Thr224Pro Gly206Asn 4 Ser207Glu 4 Trp208His 4 Thr217Gly Gly206Ser 4 Trp208Pro 4 Ala219Thr 4 Ser222Asp Ala204Thr 4 Tyr213Gln 4 Thr217Ser 4 Thr224Pro Pro205Ser 4 Thr212Ser 4 Tyr213Ile 4 Gly223Asp Gly20βGln 4 Ile209Asn 4 Thr215Pro 4 Ser220Glu Trp20δCys Thr212Gln 4 Thr215Gly 4 Ser222Glu Gly206Asn 4 Trp208Tyr 4 Ser222Glu 4 Gly223Asn Pro205Gly 4 Gly206Asn 4 Leu221Cys 4 Ser222Asp Pro205Gln + Tyr210His 4 Tyr213Ile 4 Ser220Asp Ala204His + Trp20δAsn 4 Thr212Asn 4 Ala219Asp Ala204His + Gly20βPro 4 Leu221Met 4 Gly223Asp Ala204Ser Tyr213Thr 4 Leu221Met 4 Ser222Asp Tyr213Ile + Pro214Gln 4 Ser216Glu Thr217Pro Pro205Gln 4 Tyr213Cys 4 Ser220Glu 4 Thr224Gly Gly20βAsn 4 Tyr213Asn 4 Tyr21δPro 4 Ser220Glu Pro205Ser 4 Gly206Pro 4 Tyr21δAsp 4 Gly223Pro Ala204Pro + Pro205Ser 4 Thr217Asp 4 Thr224Ser Pro205Asn Trp208Ile 4 Thr212Gln 4 Thr217Gly Ile209Cys Pro214Ser + Ser220Asp 4 Gly223Gln Ala204His Pro205Asn 4 Trp208Glu 4 Tyr210His Ile209Gly Thr215Asn 4 Thr217Asn 4 Ala219Pro Pro205Gly Pro214Asn 4 Tyr2lδlle 4 Ser222Asp Ala204Ser Pro205Ser 4 Thr212Asn 4 Tyr213Ile Pro205Gln Ser207Glu + Tyr2lOPro 4 Thr215Ser Pro205Gln Ser207Glu 4 Thr212Gln 4 Tyr213Ser Ala204Ser Ile209Asn 4 Tyr213Cys 4 Ser216Asp Trp208Ala Ile209Gln 4 Thr217Glu 4 Gly223Asn Trp2OδPro Pro214Gly 4 Tyr2IδLeu 4 Leu221Ala Gly2OβPro Thr215Ser + Ser2lβGlu 4 Leu221Cys Ala204Ser Trp208Ile 4 Ile209Val 4 Thr224Asp Tyr2lOPro Thr212Gln + Thr215Asn 4 Thr217Asp Tyr2lOSer Tyr213Cys + Pro214Asn 4 Thr217Asn Thr212Gln Tyr21δMet Ser222Asp 4 Gly223Gln Pro205Ser Trp206Met 4 Ala219Gln Leu221Asp Ala204Asn Tyr210Cys 4 Tyr213Asn 4 Tyr218Leu Ala204His Pro205Gly 4 Tyr218Ser + Ser222Asp Gly2OβPro Tyr213Asn 4 Pro214Asn 4 Tyr21δSer Ala204Pro Ile209His 4 Thr215Gln 4 Leu221Thr Ala2 04Asn Gly206Gln 4 Thr212Gly 4 Gly223Glu Thr215Gly Tyr216Ala 4 Gly223Glu 4 Thr224Pro Pro205Gln Tyr213Gly 4 Ser220Asp 4 Leu221Thr Pro205Gly Trp206His 4 Tyr2lOHis 4 Leu221Gln Tyr2lOLeu Thr212Pro 4 Tyr218Gln 4 Ala219Glu Gly2OβGln Thr212Gln 4 Tyr218His 4 Gly223Asp Trp208Ile Tyr213His 4 Thr2l5Ser 4 Ser222Glu Trp208Val Pro214Asn 4 Thr215Gly Leu221Asp Pro205Asn Tyr213Met Pro214Ser Thr224Ser Pro205Gly Trp20δMet + Ile209Leu 4 Tyr213Met Pro205Gly Thr217Glu 4 Tyr218Leu 4 Ala219His Trp208Val Tyr213Gly + Ser2lβAsp 4 Leu221Thr Tyr2lOGln Thr212Pro 4 Tyr2lδGly 4 Gly223Glu Pro205Gly Trp2OδAsp 4 Ile209His 4 Tyr210Ala Trp206Ala Ile209Leu + Tyr2lOGlu Tyr213Cys Gly206Asn Trp20δLeu 4 Tyr213Val + Ser216Asp Ala204Pro Pro205Gly Thr217Glu + Leu221Ala Trp2OδAsn Thr212Asn 4 Tyr213His 4 Ser216Glu Ala204His Thr212Gly 4 Ala219Gln 4 Leu221Thr Gly2OβGln Thr212Pro 4 Tyr213Met 4 Thr224Pro Trp208Thr Ile209Gln 4 Tyr213Ser + Ser222Asp Ala204Asn Ile209Asn 4 Tyr213Pro 4 Leu221Pro Pro205Gly Tyr210His 4 Tyr2lδlle 4 Leu221Asn Ala204Pro Tyr210Cys + Pro214Gln 4 Thr215Pro Pro205Ser Tyr213Ser 4 Ser2l^βAsp 4 Thr217Gly Pro2 05Asn Trp208Ser Ala219Glu + Ser220Asp Tyr210Ala Tyr218Met + Ala219Glu + Ser220Asp Ala204Gly Pro205Gln 4 Pro214Asp 4 Thr215Asp Tyr2lOSer Thr215Asn 4 Ser222Glu 4 Gly223Glu Gly2OβPro Tyr213Val 4 Ser222Glu 4 Gly223Asp Pro214Ser Leu221Glu + Ser222Asp 4 Gly223Gln Pro205Gln Ser216Asp 4 Thr217Glu 4 Ala219Thr

Ala204Asn - Pro205Gly -r Trp208Glu + Thr215Asn 4

Gly223Pro Pro205Gln - Gly206Pro 4 Trp208Asn 4 Thr212Gln 4

Thr224Ser Ala204Ser Pro205Ser 4 Tyr213Ser 4 Thr215Ser 4

Ala219Gly Ala204Gly 4 Gly206Gln 4 Thr212Pro 4 Ser220Asp +

Leu221Ser Pro214Ser - Thr215Gln 4 Tyr218Val 4 Ser222Asp 4

Thr224Asn Ala204Pro - Ser207A_sp 4 Tyr210Cys 4 Thr212Pro 4

Thr215Pro Gly206Asn - Pro214Gln 4 Ala219Gly 4 Gly223Asn 4

Thr224Asp Ala204Gln 4 Pro205Ser 4 Gly206Ser 4 Tyr210Gln 4

Ala219Asp Gly206Pro -<- Ser207Asp 4 Trp20δMet 4 Thr215Pro 4

Leu221Pro Ala204Asn -*- Pro205Gln 4 Tyr210Cys 4 Thr212Asn 4

Leu221Ser Ala204Asn - Gly206Gln -r Trp20δThr 4 Ile209Ser 4

Ser222Asp Pro205Gln -*- Tyr213Leu 4 Thr2:7Glu 4 Tyr216Met 4

Leu221Gln Ala204Pro + Trp208Met Ile209Val 4 Tyr210Leu 4

Trp208Ser 4 Ile209Ser 4 Tyr21δHis 4 Ala219His 4

Gly223Pro Trp20δPro + Ile209Thr 4 Thr212Gly 4 Thr215Gly +

Thr224Glu Ile209Val -r Pro214Ser 4 Ser216Asp 4 Tyr21δMet 4

Leu221Met Gly206Gln Ile209Gln 4 Ser220Asp 4 Leu221Gly 4

Thr224Ser Pro205Gln 4 Trp206Cys 4 Thr212Asn 4 Tyr213Cys +

Pro214Asp Pro205Gln + Trp208Gln 4 Thr217Asp 4 Gly223Ser 4

Thr224Gly Ser207Glu + Thr212Ser 4 Thr217Gly + Tyr218Gln 4

Leu221His Tyr210His 4 Thr212Ser 4 Leu221Glu 4 Gly223Ser 4

Thr224Pro Trp20δPhe 4 Tyr213Met 4 Thr215Pro 4 Ser220Glu 4

Thr224Gly Gly206Ser 4 Trp20δPhe 4 Ile209Ser 4 Tyr213Cys 4

Ser216Glu Ala204Gln + Pro205Ser 4 Tyr210His 4 Tyr21δPro 4

Thr224Glu Pro205Gln + Gly206Gln 4 Leu221His 4 Ser222Asp +

Gly223Gln Ala204Gly 4 Ile209His 4 Tyr210Met 4 Ser216Glu 4

Thr217Pro Gly206Pro ^■>^■ Ile209Ala t Tyr210Gly + Leu221Asp 4 Gly223Gln Gly206Asn - Tyr210Met 4 Thr215Glu 4 Gly223Asn 4

Thr224Gly Gly206Gln 4 lle209Pro 4 Ser216Asp 4 Leu221Cys 4

Gly223Pro Trp20δThr -r Tyr210Val 4 Thr212Ser 4 Pro214Asp 4

Gly223Gln Thr212Gly τ Pro2:4G:n 4 Thr2:5Ser 4 Leu22:Gly 4

Thr224Glu A_la204Thr 4 Thr212Ser -r Thr217Ser 4 Gly223Asp +

Thr224Pro Pro205Ser 4 Ile209Pro 4 Tyr213Ile 4 Ala219Pro 4

Ser222Glu Trp208Gly + Thr212Gln 4 Tyr216Gly 4 Ser222Glu 4

Gly223Ser Gly206Pro + Ile209Pro 4 Tyr213Ser 4 Thr217Gly 4

Tyr218Val Pro205Gln - Tyr213Cys 4 Pro214Gln 4 Ser220Glu +

Thr224Gly Gly206Asn - Thr212Gly Tyr213Asn 4 Tyr218Pro 4

Ser220Glu Ala204Pro 4 Pro205Ser 4 Thr212Gln 4 Thr217Asp 4

Thr224Ser Pro205Asn 4 Trp20δlle 4 Thr212Gln 4 Pro214Ser 4

Thr217Gly Ile209Cys 4 Thr217Gly 4 Ser220Asp 4 Leu221Gln 4

Gly223Gln Ile209Thr 4 Thr215Gly + Ser216Glu 4 Tyr216Leu 4

Ala219Pro Ile209Met 4 Pro214Gly 4 Thr217Gly 4 Tyr216Cys 4

Leu221Met Pro205Gly 4 Pro214Asn 4 Tyr21δlle + Ala219Gly 4

Ser222Asp Tyr210Thr + Thr212Ser 4 Pro214Ser 4 Leu221Asn 4

Ser222Asp Gly206Asn 4 Tyr210Ile 4 Pro214Gln 4 Ala219Asn 4

Leu221Val Pro205Asn 4 Thr215Ser 4 Ala219Ser 4 Leu221Ala 4

Thr224Asp Gly206Pro 4 Pro214Asn 4 Thr215Gln 4 Tyr21δSer 4

Leu221Thr Ala204Pro 4 Gly206Gln 4 Ile209Hιs 4 Thr212Gly 4

Gly223Glu Pro205Gln 4 Tyr213Gly 4 Tyr21δAla 4 Gly223Glu 4

Thr224Pro Pro205Ser 4 Pro214Gly 4 Thr217Pro 4 Ala219Thr 4

Leu221Glu Ala204Pro 4 Pro205Asn 4 Ser207Asp 4 Tyr210Ala 4

Pro214Ser Gly206Ser 4 Tyr213Thr 4 Ala219Gln 4 Ser220Glu 4

Gly223Ser Pro205Gly 4 Thr212Asn 4 Thr215Pro 4 Ser216Glu 4

Leu221Asn Ser207Glu - He209Ala 4 Thr212Asn 4 Thr215Gln 4

Tyr218Me_ Tyr213His -*^■ Thr215Gln 4 Thr217Asn 4 Ala219Glu 4

Leu221Ser Glv20SGin - Tyr213Asn 4 Thr215Ser + Tyr218Ala +

Leu221Ala Trp208Met Tyr210Met 4 Tyr218Gln + Ser220Glu 4

Thr224Ser Ala204Thr Pro205Ser 4 Ile209Leu 4 Thr217Asn 4

Thr224Pro Pro205Gly -^»- Ile209Leu 4 Thr215Asn 4 Ser216Glu +

Gly223Ser lle209Ser 4 Tyr213Leu 4 Thr217Asp 4 Tyr21δAsn +

Ala219His Ala204Ser 4 Pro205Ser 4 Ser207Glu 4 Leu221Val Ala204His + 4 Leu221Thr 4

Pro205Ser 4 Gly206Gln 4 Thr212Pro 4 Gly223Pro 4

Thr224Pro Ala204Asn 4 Trp206Thr 4 Tyr213Pro 4 Leu221Pro +

Ser222Asp Pro205Gly + Ile209Met 4 Tyr216Met 4 Leu221Val +

Gly223Asn Thr212G:y Tyr216Ile 4 Ala219Asn 4 Ser220Glu 4

Thr224Pro Ala204Gln 4 Pro205Gln 4 Gly206Gln -<- Ile209Met 4

Ser216Asp Ala204Thr 4 Thr212Gly 4 Ala219Asn 4 Gly223Asp 4

Thr224Asn Ala204Pro 4 Trp20δHis 4 Tyr213Leu 4 Thr217Ser 4

Ser222Glu Pro205Ser 4 Ser207Asp 4 Ile209Asn 4 Tyr213Met +

Thr217Gln Gly206Pro + Trp20δSer 4 Thr212Gly + Tyr213Cys 4

Gly223Glu Ala204Asn 4 Gly206Pro 4 Trp206Val 4 Pro214Asn +

Tyr218Glu Ile209Gly 4 Tyr210Ala + Tyr218Pro 4 Gly223Ser +

Thr224Ser Pro205Gln 4 Tyr213His Tyr218Met Ser220Glu +

Leu221Gly Ala204Asn 4 Tyr210Cys 4 Thr212Gln 4 Tyr21δLeu 4

Ser222Asp Gly206Ser 4 Ile209Leu 4 Thr212Gly 4 Pro214Gln 4

Thr215Glu Ser207Asp 4 Pro214Ser 4 Thr215Asn 4 Thr217Asn 4

Leu221Ser Pro205Gly 4 Trp206Thr 4 Thr217Gly 4 Ser220Glu +

Gly223Ser Gly206Ser Trp20δGlu 4 Tyr210Gln + Thr217Gln +

Leu221Pro Gly206Gln 4 Ser207Glu 4 Thr212Asn 4 Pro214Ser 4 Leu221Ala Ala2_.4Ser ^ Trp203Leu 4 He209Asn 4 Thr215Asp 4

Thr224Asn Gly206Ser + Ile209Pro 4 Thr217Ser 4 Ser220Glu 4

Gly223Gln Pro205Gln 4 Trp206Pro 4 Thr212Pro 4 Pro214Glu 4

Ala219Ser Gly206Pro 4 Ser207Glu 4 Tyr210Met 4 Thr212Gly 4

Tyr213Leu Ala204Hιs 4 Tvr210Pro 4 Tyr213Ala 4 Thr217Glu 4

Thr224Ser Trp2C8Cys + Ile209Met -r Tyr210Ala + Tyr213Me_ ~

Ala219Asn Trp208Thr + Tyr210Ala + Tyr218Met 4 Ala219Gl_ 4

Ser220Asp Trp208Asn -t Ile209Pro 4 Ala219Asp + Ser220Asp 4

Gly223Ser Pro205Ser 4 Tyr213Thr 4 Pro214Asp + Thr215Asp 4

Tyr218Val Ile209Ser 4 Tyr213Pro 4 Leu221Met + Ser222Asp 4

Gly223Asp Ala204Thr 4 Thr217Gln 4 Tyr218Ile 4 Ser222Glu 4

Gly223Asp Pro205Ser + Tyr210Ala 4 Thr215Asn 4 Ser222Glu 4

Gly223Glu Tyr210Pro 4 Thr212Pro 4 Leu221Asn 4 Ser222Asp 4

Gly223Asp Trp206Pro 4 Ile209Pro 4 Tyr213Ile 4 Ser222Asp 4

Gly223Asp Gly206Gln 4 Tyr213Gly 4 Thr217Gly 4 Ser222Glu 4

Gly223Glu Trp208Ser 4 Ile209Pro 4 Thr212Pro 4 Ser216Glu 4

Thr217Asp Gly206Asn 4 Tyr218Met 4 Ala219His 4 Ser220Glu 4

Leu221Asp Ile209Ala + Thr215Glu 4 Ser216Glu 4 Gly223Gln 4

Thr224Asn Ile209Gly + Pro214Ser 4 Thr215Glu + Ser216Asp 4

Tyr21δlle Ala204Ser 4 Trp206Val 4 Ile209Ser 4 Thr215Glu 4

Ser216Asp Thr212Pro + Thr215Asp 4 Ser21βGlu 4 Thr217Glu 4

Thr224Ser Ala204Hιs 4 Ser207Glu 4 Trp20δHis 4 Tyr210His 4

Ser222Asp Ser207Glu 4 Tyr213Asn 4 Leu221Thr 4 Ser222Asp 4

Thr224Gly Gly206Ser 4 Ser207Glu Trp20δPro + Ala219Thr 4

Ser222Asp Pro205Gly + Ser207Glu 4 Thr212Gln + Tyr213Gln 4

Ser222Asp Pro205Ser 4 Ser207Glu 4 Tyr213His Thr217Pro 4

Ser222Glu Gly206Pro + Ser207Asp 4 Tyr21311e t Tyr21δAsn 4

Ser222Glu Gly206Pro - Trp206Glu 4 Tyr210Cys 4 Pro214Gln 4

Ser220Asp Tyr210Glu - Tyr213Gly 4 Tyr216Leu 4 Ala219Asp 4

Thr224Ser Gly206Pro ÷ Ser207Asp 4 Leu221Asp 4 Ser222Glu +

Thr224Gln Tyr213Ser -*- Pro214Ser 4 Thr215Glu 4 Thr217Asp +

Tyr21δAla Ala204Asn ^■+^■ Tyr210Gln 4 Thr212Asn 4 Pro214Glu +

Ser216Asp Pro205Gln -*- Gly206Pro 4 Pro214Glu 4 Ser216Asp +

Thr224Ser Ser207Asp + Thr212Pro 4 Thr215Gly 4 Thr217Gly +

Gly223Asp Ser207Glu 4 Tyr210Val 4 Pro214Asn 4 Leu221Thr 4

Gly223Glu Ser207Asp - Tle209Thr 4 Tyr210Met + Thr212Pro +

Gly223Asp Ser207Glu -*- Tyr210Ser 4 Tyr21δLeu 4 Ser220Asp +

Ser222Glu Ser207Asp 4 Thr217Gln 4 Ser220Asp 4 Ser222Asp +

Thr224Asn Trp20δThr 4 Thr217Glu 4 Tyr21δSer 4 Ala219Glu 4

Leu221Cys Pro205Gly 4 Thr212Ser 4 Ala219Gln + Ser220Glu +

Ser222Glu Gly206Pro + Trp206His 4 Tyr210Gly 4 Ser220Glu 4

Ser222Asp Ala204Gln 4 Gly206Ser 4 Trp20δGln 4 Ser220Glu +

Ser222Glu Ala204Ser Tyr213Thr 4 Ser220Asp + Leu221Met +

Ser222Asp Thr212Gly + Thr215Asp 4 Ser216Asp 4 Thr217Gly +

Tyr21δAsp Ala204Asn 4 Tyr210Glu 4 Tyr213Ala 4 Ser220Glu +

Ser222Glu Ala204His 4 Gly206Pro 4 Ser216Glu 4 Tyr21δAsp +

Leu221Ser Gly206Pro 4 Ile209Ser 4 Tyr210His 4 Ser222Glu +

Thr224Asp Trp208Met + Tyr218Leu 4 Ala219Pro 4 Ser222Glu +

Thr224Glu Trp20δVal 4 Tyr210Leu + Thr212Asn 4 Tyr216Asp +

Ser220Glu Ala204Ser 4 Tyr213Gln 4 Tyr216Asp 4 Ala219Pro +

Ser220Glu Thr212Asn 4 Tyr213Gln 4 Thr217Ser 4 Tyr218Glu +

Ser220Glu Pro214Ser 4 Leu221Glu 4 Ser222Asp 4 Gly223Ser 4

Thr224Asp Pro205Gly + Tyr210Cys 4 Ala219Asp 4 Ser220Glu 4 Ser222Asp

Gly206Gln 4 Tyr210Gly + Pro214Glu 4 Thr217Pro 4

Tyr218Glu Trp208Asp 4 Pro214Ser 4 Thr217Gln 4 Ser220Asp +

Gly223Asp Ser207Glu 4 Tyr210Ala 4 Thr215Asn + Ser220Asp +

Leu221Ala Ser207Asp 4 Trp206Cys 4 Ile209Ala 4 Thr215Asn 4

Ser220Glu Ser207Asp + Tyr210Met Tyr218Asn 4 Ser220Glu 4

Leu221Cys Ser207Glu 4 Tyr210Pro 4 Thr215Gln 4 Tyr218Cys 4

Ser220Glu Ser207Glu 4 Thr212Ser 4 Pro214Gln 4 Ser220Asp +

Leu221Pro Pro205Asn + Ser207Asp + Thr215Pro + Ser220Asp +

Leu221Thr Trp208Glu + Ile209Asn 4 Thr217Gly + Ala219Asp +

Ser222Glu Pro205Asn 4 Ser207Glu 4 Tyr213Leu 4 Ser220Asp +

Gly223Glu Ala204Ser 4 Pro205Gly 4 Ser207Asp 4 Thr212Ser 4

Thr224Asp Ala204Pro 4 Gly206Gln + Ser207Glu 4 Tyr216Asn 4

Thr224Glu Gly205Ser 4 Ser207Glu 4 Tyr210Met 4 Tyr213Cys 4

Thr224Asp Pro205Asn 4 Ser207Glu 4 Ile209Val 4 Tyr213Pro 4

Thr224Glu Ser207Asp 4 Ile209Val 4 Tyr213Ala 4 Gly223Gln 4

Thr224Glu Pro205Asn 4 Ser207Glu 4 Ala219Glu 4 Ser220Glu +

Gly223Gln Gly206Asn 4 Ser207Glu 4 Trp206Asp 4 Ala219Asp 4

Thr224Pro Ala204Gly 4 Gly206Ser 4 Ser207Asp 4 Tyr210Asp 4

Leu221Gly Ala204Pro 4 Ser207Glu 4 He209Thr 4 Tyr210Glu +

Tyr218Leu Ile209Thr 4 Tyr213Gly + Ala219Asp 4 Ser222Glu +

Gly223Asp Pro205Gln 4 Tyr213Gln + Tyr218Glu 4 Leu221Glu +

Ser222Asp Gly206Gln 4 Ser207Glu 4 Thr212Asn 4 Ser220Glu 4

Thr224Glu Ser207Glu + Thr217Gly 4 Tyr21δAsn 4 Ser220Asp 4

Thr224Glu _Ala204Thr 4 Tyr210Cys 4 Ser220Glu 4 Leu221Ser 4

Gly223Glu Ile209Pro 4 Ser216Glu 4 Ala219Glu 4 Ser220Asp 4

Gly223Asn Gly20βGln 4 Tyr210Met 4 Thr212Pro 4 Ala219Glu +

Ser222Asp ?ro205Asn - Tyr210Glu 4 Thr217Asp 4 Tyr216Ala 4

Thr224Pro Ala204Gly - Trp208Gln 4 Thr212Gln 4 Ser216Asp 4

Ala219Glu Ala204Asπ -r Trp20δAla + Ser216Asp 4 Ala219Glu 4

Thr224Gly Ala204Asn Tyr210Asp 4 Thr215Glu 4 Thr217Glu 4

Ala219Gly Trp20δLeu -^•- Ile209Gly 4 Thr215Asp 4 Thr217Glu 4

Ser220Asp Thr212Pro - Tyr213Leu 4 Ser216Glu 4 Tyr218Glu 4

Leu221Glu Thr217Asp 4 Ala219Glu 4 Leu221Asn 4 Ser222Glu 4

Thr224Ser Pro205Ser + Trp208Glu 4 Tyr218Glu + Leu221Gly +

Ser222Glu Ser207Glu 4 Thr215Pro Thr217Asp 4 Ser220Glu 4

Gly223Pro Thr217Asp Ala219Asn 4 Leu221Glu 4 Ser222Glu 4

Gly223Asn Trp208Tyr 4 He209Asn 4 Thr217Glu + Leu221Glu 4

Ser222Asp A_la204Gly 4 Tyr210Glu 4 Pro214Glu 4 Ser216Glu 4

Thr217Asn Pro214A-sp 4 Thr215Gln 4 Ser216Glu 4 Ser220Glu +

Leu221Met Ala204Gln Tyr210Glu 4 Thr212Asn Thr217Glu 4

Ser222Asp Trp208Leu 4 Ile209Val 4 Thr215Asp 4 Thr217Glu 4

Leu221Glu Gly206Pro 4 Ile209Val 4 Tyr213Pro 4 Thr215Asp

Ala219Glu Pro205Asn 4 Ser207Glu 4 Tyr218Asp 4 Ser222Asp 4

Thr224Gly Gly206Ser 4 Tyr210Asp Pro214Asp 4 Thr215Pro 4

Leu221Asp Tyr210Ser 4 Pro214Asp 4 Tyr21δCys 4 Ser220Glu 4

Ser222Glu Ser207Asp 4 Ile209Ser 4 Thr212Ser 4 Pro214Asp 4

Thr215Asp

TABLE27

Loop 6 - Sextuple Substitution Variants

Gly206Asn 4 Tyr210Gly + Thr212Gln 4 Tyr213Gly 4

Tyr218Ile + Thr224Gly Pro205Asn 4 Gly206Ser Tyr213Gly 4 Ser216Asp +

Thr217Ser Thr224Asn Ala204Ser Pro205Ser 4 Tyr213Ser 4 Tyr218His 4

Ala219Gln Leu221Asn Ser207Glu 4 Thr212Gln 4 Thr215Pro 4 Tyr218Gln 4

Leu221Met Gly223Gln Pro205Ser 4 Ser2C7Asp -r Tyr210Val 4 Tyr213Leu 4

Leu221Gln 4 Thr224Gly Ala204Ser 4 Trp208Cys 4 lle209His 4 Thr215Glu 4

Thr217Pro + Gly223Ser Ala204Gly 4 Ile20^*9Asn Tyr210Ile 4 Ser216Asp 4

Ala219Asn 4 Leu221Met Ala204Gly Pro205Gln Pro214Glu 4 Thr215Ser 4

Tyr218Asn 4 Thr224Ser Gly206Asn 4 Trp208Cys 4 Thr212Gly 4 Tyr213Gly 4

Ser216Asp 4 Leu221His Trp206Pro 4 He209Gly 4 Thr215Ser 4 Ala219Thr 4

Leu22:Thr 4 Thr224Asp Pro205Gly 4 Gly206Gln 4 Ile209Gly + Thr212Asn +

Pro214Asn 4 Ser220Glu Pro205Gly 4 Trp20δLeu 4 Thr217Gln 4 Leu221Gln

Gly223Gln Thr224Glu Ala204Gly -t- Gly206Asn 4 Tyr210Cys 4 Tyr213Thr 4

Gly223Asp 4 Thr224Asn Gly20βAsn 4 Trp208Hιs + Ile209Gly ^■*- Thr217Gly 4

Leu221Val 4 Thr224Pro Ala204Thr 4 Thr212Gln 4 Tyr213Gln 4 Thr217Ser 4

Ser222Asp 4 Thr224Pro Trp206Gly 4 Thr212Gln + Tyr213Leu 4 Tyr21δGly 4

Ser222Glu 4 Gly223Ser Pro205Ser 4 Trp208Ser 4 Tyr213Asn 4 Tyr218Gln 4

Ser222Asp 4 Thr224Gln Ala204His 4 Pro205Gly 4 Ile209Asn 4 Tyr213Gln 4

Thr215Ser 4 Thr217Ser Ala204Thr 4 Gly206Asn 4 Trp206Leu 4 Thr215Ser 4

Ser216Asp Ala219His Gly206Ser 4 Thr212Gln 4 Tyr213Val + Tyr216Cys 4

Ser220Glu + Thr224Gln Gly206Asn 4 Trp208Ile + lle209Ser 4 Tyr216Gln 4

Gly223Glu + Thr224Gly Gly206Asn 4 Ser207Glu 4 Ile209Cys 4 Thr212Ser 4

Thr217Pro + Ala219His Ala204Asn 4 Tyr210Cys + Tyr213Asn Tyr21δLeu +

Leu221His 4 Ser222Glu Gly206Pro 4 Tyr213Asn Pro214Asn 4 Thr215Gln +

Tyr21δSer 4 Leu221Thr Ala204Pro 4 Pro205Asn 4 Gly206Gln 4 Ile209His 4

Thr212Gly 4 Gly223Glu Pro205Gln 4 Tyr213Gly + Thr215Gly 4 Tyr21δAla 4

Gly223Glu 4 Thr224Pro Ala204His 4 Ser207Glu 4 Trp20δGly + Ile209His 4

Tyr213Cys 4 Thr215Ser Gly206Ser 4 Trp208His + Tyr213Thr + Ala219Gln +

Ser220Glu + Gly223Ser Tyr213His Thr215Gln 4 Thr217Asn 4 Tyr216Ala +

Ala219Glu Leu221Ser Ala204Asn + Pro205Ser 4 Gly206Asn 4 Ile209Leu +

Thr215Asp 4 Thr224Pro Pro205Ser 4 Ile209Gly 4 Tyr213Leu 4 Thr217Asp 4 Tyr218Asn + Ala219His Ala204Asn 4 Pro205Gln + Ser207Asp + Tyr210Val 4

Thr212Gly + Leu221Thr Ala204Asn 4 Trp208Thr 4 Ile209Gln + Tyr213Pro 4

Leu221Pro + 3er222Asp Ala204Ser ⁺ Gly206Ser + Tyr213Gly Pro214Gln 4

Tyr218Val 4 Ser222Asp Ala204Pro 4 Tyr210Cys 4 Tyr213Ser 4 Pro214Gln 4

Thr215Pro 4 Thr217Gly Thr212Ser 4 Pro214Asp Thr217Gly Ala219Thr 4

Leu221Pro 4 Thr224Gln Pro205Gln 4 Gly206Gln 4 Ser207Glu 4 Tyr210Gly 4

Thr212Pro 4 Tyr218Gly Pro205Gln 4 Gly206Pro 4 Trp208Asn 4 Ile209Met 4

Tyr213Ser 4 Ala219Asp Ala204Asn 4 Ser207Glu + Trp206Ala 4 Tyr213Asn 4

Leu221Thr 4 Thr224Gly Ala204Pro 4 Trp20δHis 4 Ile209Asn 4 Tyr213Leu 4

Thr217Ser 4 Ser222Glu Gly206Pro + Trp206Ser 4 Tyr210Val 4 Thr212Gly 4

Tyr213Cys 4 Ala219Asp Pro205Gly 4 Gly206Gln + Ile209Cys 4 Tyr210His 4

Thr215Gln 4 Ala219Thr Trp208Gly + Ile209Ser + Thr212Gln 4 Tyr216Met 4

Leu221Ser 4 Gly223Ser Pro205Asn 4 Ile209Asn 4 Thr212Asn 4 Thr217Glu 4

Gly223Pro 4 Thr224Ser Pro205Gly 4 Trp20δThr 4 Thr212Ser 4 Ser216Glu 4

Thr217Gly 4 Gly223Ser Gly206Ser 4 Trp20δGlu 4 Tyr210Gln 4 Thr215Gly 4

Thr217Gln 4 Leu221Pro Gly206Ser + Ser207Glu 4 Trp20δLeu 4 Thr212Asn 4

Leu221Ala 4 Thr224Asn Ala204Asn 4 Pro205Gln 4 Gly206Gln 4 Trp20δlle 4

Ser2lβAsp Tyr21δCys Ala204Thr 4 Tyr210Cys 4 Tyr213Ser 4 Thr215Ser

Ser2lβAsp Ala219His Trp20δLeu + Ile209Asn 4 Thr212Gly 4 Thr215Ser +

Tyr218Pro 4 Leu221Gly Pro205Gly + Gly206Gln 4 Tyr216Gly 4 Ala219Asn 4

Leu221Gln + Thr224Ser Pro205Gln + Trp208Val 4 Tyr210Pro 4 Thr215Gly 4

Ala219Ser Thr224Glu Pro205Gly Trp208Met + Ile209Ser 4 Thr212Asn

Tyr218Ala 4 Ala219Thr Thr212Gly + Tyr213Asn 4 Pro214Ser 4 Ser216Glu 4

Thr217Pro 4 Leu221Thr Ala204Gly 4 Trp208Pro Ile209Pro 4 Tyr213Gln 4

Tyr21δSer 4 Ser220Glu Ala204Gly 4 Gly20βAsn 4 Ile209Met 4 Thr212Pro 4

Ala219Glu 4 Leu221Met Pro205Asn 4 Gly206Pro 4 Trp20δLeu 4 Tyr210Pro 4

Thr217Gln 4 ^"τyr218Glu Pro205Asn - Gly206Gln 4 Tyr213Val 4 Ser220Asp 4

Gly223Ser 4 Thr224Asn Aia204Thr -r Pro205Gly + Gly206Gln 4 Pro214Gln 4

Leu221Val + Gly223Gln Pro2053er ÷ Ile209Gln 4 Thr212Pro 4 Ala219Gly 4

Ser220Glu 4 Gly223Gln Ala204Gly 4 Gly206Gln 4 Ile209Leu 4 Thr212Asn 4

Pro214Gln 4 Thr215Ser Gly206Asn 4 Trp208Ser 4 Thr212Ser 4 Pro214Asn 4

Ser220Glu 4 Gly223Ser Ala204His + Tyr213Leu 4 Pro214Asn 4 Thr217Pro 4

Ser220Glu 4 Gly223Gln Gly206Pro + Ile209Cys Pro214Gln 4 Thr217Ser 4

Tyr218Cys 4 Ala219Gln A_ia204Thr + Tyr210Ala Thr215Gly 4 Thr217Gln 4

Ala219Pro 4 Ser220Asp Ser207Asp + Ile209Thr 4 Thr212Gly 4 Thr217Pro 4

Ala219Gly 4 Thr224Pro Ala204Gly 4 Ser207Glu + Thr212Asn 4 Pro214Ser 4

Tyr216Ser + Ala219Ser Pro205Gln + Gly206Gln 4 Thr212Ser 4 Tyr213Ala 4

Thr215Ser 4 Gly223Glu Pro205Ser + Tyr210Ala 4 Pro214Ser 4 Thr215Asn 4

Ser222Asp 4 Gly223Glu ϊle209Pro 4 Tyr210Pro 4 Thr212Pro 4 Leu221Asn 4

Ser222Asp 4 Gly223Asp Pro205Ser + Thr212Ser 4 Tyr213Ile 4 Ala219Pro 4

Ser222Glu Gly223Asp Ala204Asn 4 Ile209Cys 4 Tyr213Cys 4 Tyr218Ala 4

Leu221Asp + Ser222Glu lle209Val + Thr212Asn Tyr213Met 4 Thr217Asn 4

Gly223Asp 4 Thr224Asp Pro205Gly + Ile209Asn Thr212Asn 4 Ser216Glu 4

Thr217Glu 4 Tyr218Gln Ala204His 4 Trp208Ser 4 Thr212Pro 4 Tyr213Pro 4

Ser21βGlu 4 Thr217Asp Pro205Ser + Ile209Ala + Thr212Gly 4 Ser216Asp 4

Thr217Glu 4 Ala219Thr Pro205Ser + Trp208Met 4 Pro214Asn 4 Ala219Gln 4

Ser220Glu Leu221Asp Ala204Pro + Gly206Ser 4 Tyr213Ile Ser220Glu 4

Leu221Asp + Gly223Ser Gly206Pro + Tyr210Gly + Tyr213His 4 Thr217Glu 4

Tyr21δAsp + Leu221Asn Thr212Pro + Tyr213Ser 4 Thr215Asp 4 Ser216Asp 4

Tyr21δPro 4 Gly223Gln Ser207Glu + Ile209Asn 4 Thr212Ser 4 Thr217Gly 4

Ser222Glu 4 Thr224Gln Pro205Gly Ser207Glu 4 Tyr210Ser 4 Pro214Gln 4

Ser222Asp + Gly223Asn Gly206Pro 4 Ser207Asp 4 Trp208Ser + Pro214Ser 4

Leu221Asn 4 Ser222Asp Gly206Gln + Ser207Asp 4 Ile209Cys 4 Tyr210Met 4 Thr217Asn -r Ser222Glu Gly206Pro 4 Ser207Glu + Ile209Pro -r Tyr210Ile 4

Leu221Asn 4 Ser222Glu Pro205Ser 4 Ser207Glu 4 Trp208Hιs 4 Thr215Pro 4

Leu221Asn + Ser222Glu Pro205Asn 4 Ser207Asp + Thr215Gln 4 Tyr21δlle 4

Leu221Gly ^ Ser222Glu Ala204Pro 4^" Ser207Asp 4 Tyr210Cys + Thr212Pro 4

Thr215Pro 4 5er222Glu Pro205Gly 4 Pro214Asp 4 Thr215Glu 4 Ser216Asp -*-

Thr217Pro 4 Gly223Gln Ile209Thr 4 Thr217Gly + Ala219Glu 4 Ser220Asp +

Leu221Glu 4 Gly223Ser Ala204Gln 4 Ser207Asp + Ile209Thr 4 Tyr216Asn +

Ser222Glu 4 Gly223Asp Pro205Ser + Ser207Glu 4 Pro214Gly 4 Thr215Asn 4

Ser222Asp 4 Gly223Glu Pro205Asn 4 Gly206Pro -t Trp208Asp 4 Thr212Gln -r

Ser222Asp 4 Gly223Ser Gly206Gln + Pro214Asp 4 Thr215Asp 4 Thr217Asp 4

Gly223Ser *^■ Thr224Gly Ala204Hιs 4 Ile209Thr 4 Ser216Asp 4 Thr217Asp 4

Tyr21δAsp 4 Leu221Ser Thr212Asn 4 Pro214Gln 4 Tyr21δMet 4 Ser222Glu +

Gly223Asp 4 Thr224Glu Ala204Gln 4 Gly206Pro 4 Tyr210Glu 4 Pro214Gly

Ala219Asp 4 Leu221Gln Ala204Gly + Pro205Gln 4 Trp2OδAsp 4 Ile209Gln 4

Pro214Gly 4 Leu221Glu Ala204Asn 4 Gly206Gln 4 Ser207Glu 4 Trp20δHis 4

Leu221Asp 4 Ser222Asp Trp206Gly + Tyr213Ser 4 Pro214Ser Thr215Glu +

Thr217Asp 4 Tyr21δAla Ala204Asn + Pro205Ser + Tyr210Pro 4 Pro214Asp +

Ser216Glu 4 Tyr21δlle Gly20βGln 4 Ile209Leu 4 Thr212Gly 4 Pro214Asp +

Ser216Glu 4 Leu221His Pro205Gln + Trp208Glu 4 Tyr210Asp 4 Thr212Gln 4

Tyr213Asn 4 Thr215Ser Ser207Asp 4 Thr212Pro 4 Thr215Gly 4 Thr217Gln 4

Leu221Met 4 Gly223Asp Ala204Asn 4 Pro205Gln 4 Ser207Glu 4 Trp206Met 4

Ala219Pro 4 Gly223Asp Ala204Thr 4 Ser207Asp + Thr212Gly 4 Ala219Asn 4

Gly223Asp 4 Thr224Asn Ser207Asp Pro214Asn 4 Thr215Gly 4 Tyr21δAla +

Leu221Ser 4 Gly223Asp Ser207Glu 4 Thr212Asn + Tyr213Gln 4 Tyr216Pro 4

Ser220Glu 4 Leu221Asp Ala204Asn Gly206Ser 4 Ser207Asp 4 Tyr210Thr 4

Ser220Asp 4 Leu221Asp Gly206Ser + Thr212Asn 4 Thr215Pro 4 Tyr21δVal +

Leu221Asp 4 Gly223Asp Trp20δMet -r Tyr210Thr 4 Thr212Asn 4 Ala219Gln 4

Leu221Asp 4 Gly223Asp Gly20βSer -*^■ Ser207Glu -r Pro214Ser Ala219Asn +

Ser220Glu - Ser222Glu Ala204Hιs - Ser207Glu - Ala219Ser - Ser220Glu

Leu221H s -*- Ser222Asp Pro2G5Ser 4 Ser207G:u 4 Tyr2:3His 4 Thr2:7Pro 4

Ser220Glu 4 Ser222Glu 5er207Glu 4 Ile209Met Tyr210Gly 4 Tyr213Leu 4

Ser220Glu 4 Ser222Asp 5er207Asp Tyr213Pro Pro214Gln 4 Ser220Glu 4

Ser222Glu -r Gly223Pro Gly206Ser 4 Trp2θ^"δGln 4 Tyr210Glu 4 Thr212Asn 4

Thr215Gly + Leu221Glu Pro205Gly - Thr212Ser 4 Tyr21δLeu + Ala219Gln +

Ser220^"Glu -*^■ Ser222Glu Trp208Phe -t Thr212Asn 4 Pro214Ser 4 Ser220Glu 4

Ser222Asp 4 Gly223Gln Ala204Gly 4 Gly206Pro 4 Tyr210Ser 4 Tyr218Cys 4

Ser220Glu 4 Ser222Asp Pro205Ser 4 Trp20δLeu 4 Tyr213Ala 4 Thr217Ser 4

Ser220Asp 4 Ser222Asp Pro205Gly 4 Trp20δHis 4 Tyr210His 4 Ser220Glu 4

Leu221Gln 4 Ser222Asp Pro205Gly + Thr212Gln 4 Ala219Thr -^ Ser220Glu +

Ser222Asp + Gly223Pro Ala204Ser -r Gly20^~6Pro + Tyr210Gly 4 Ser220Glu 4

Leu221Ser 4 Ser222Asp lle209Ser 4 Tyr210Ser 4 Thr212Asn 4 Tyr213Cys 4

Ser220Asp 4 Ser222Glu Ala204Asn 4 Pro205Gly 4 Ser207Asp 4 He209Ser 4

Tyr213Val 4 Leu221Glu Ala2^~04Asn 4 Gly206Pro 4 Thr215Glu 4 Ser216Glu 4

Thr217A_sn Tyr21δGlu Gly206Pro 4 Ile209Ser 4 Tyr210His 4 Thr217Gly 4

Ser222Glu 4 Thr224Asp Gly206Gln 4 Thr217Gly 4 Ser220Asp 4 Ser222Glu 4

Gly223Glu 4 Thr224Ser Gly206Gln 4 Trp206Gln 4 Ile209Gln 4 Ser216Asp 4

Tyr216Asp 4 Ala219Glu Gly206Asn 4 Thr212Pro 4 Pro214Ser 4 Thr217Glu 4

Tyr216Glu 4 Ser220Glu Gly206Gln 4 Trp20δSer 4 Tyr210Asp 4 Pro214Gln 4

Thr217Glu 4 Ala219Asp Gly206Ser 4 Ser207Glu 4 Trp20δPro 4 Tyr210Asp 4

Ala219Thr 4 Ser222Asp Trp206Gly 4 Tyr210His Thr212Ser 4 Pro214Glu 4

Tyr218Asp 4 Leu221Ile Ala204Gln 4 Gly206Gln 4 Tyr210Gly -t Pro214Glu 4

Thr217Pro 4 Tyr218Glu Ala204His 4 Pro214Glu + Thr215Gln 4 Thr217Gln 4

Tyr218Glu 4 Leu221Pro Ser207Asp 4 Ile209Cys 4 Thr215Pro Tyr21δHis 4 Ala219Thr - Ser220Glu Pro205Asn - Ser207Glu 4 Trp206Tyr 4 Ile209Asn 4

ThrlllGln ~ Ser220Glu Ser207Asp 4 Pro214Ser 4 Thr215Asn + Thr217Asn 4

5er220Giu + Leu221Ser Pro205Gly 4 Ser207Asp + Tyr210Ser 4 Ser220Asp 4

Gly223Pro + Thr224Ser Gly206Gln 4 Ser207Glu + Ile209Gln 4 Ser220Asp +

Leu221Ala 4 Thr224Ser Ala204Ser 4 Pro205Gly 4 Ser207Asp 4 Thr212Ser 4

Thr215Gln + Ser220Glu ?ro205Ser 4 Ser207Asp 4 Thr217Gln 4 Ala219His +

Ser220Glu -r Gly223Glu 3er207Glu 4 Thr215Gln 4 Tyr216Val 4 Ser220Asp 4

Gly223Glu 4 Thr224Asn Pro205Gly 4 Ile209Pro 4 Tyr210Asp 4 Ala219Asp

Ser222Asp 4 Gly223Ser Gly206Asn 4 Tyr210Gln + Pro214Asn 4 Thr215Glu +

Tyr216Glu 4 Leu221Ala Ile209Asn 4 Tyr210Asp 4 Thr212Gln 4 Ser222Glu 4

Gly223Pro 4 Thr224Asn Ala204Gly 4 Gly206Gln 4 Ser220Asp 4 Leu221Asp

Gly223Asn + Thr224Glu Gly206Pro 4 Thr212Pro 4 Thr217Pro + Tyr216Asn 4

Leu221Glu 4 Thr224Glu Ile209His 4 Tyr210Leu + Tyr213Leu 4 Pro214Asp 4

Thr217Asp + Ser220Glu Ile209Asn 4 Tyr210Ile + Thr212Asn 4 Thr215Gly 4

Ser220Asp 4 Gly223Glu Gly206Gln + Tyr210Ala + Thr215Asn 4 Ser216Glu +

Thr217Asp Ser220Asp Trp206Thr 4 Tyr213Pro 4 Tyr216His 4 Ala219Glu +

Leu221Cys 4 Ser222Asp Ala204Gly Trp20δGln 4 Thr212Gln 4 Ser216Asp +

Ala219Glu 4 Gly223Ser Gly206Ser 4 Pro214Gly 4 Ser216Asp 4 Tyr216Pro 4

Ala219Glu 4 Leu221Pro Pro205Asn 4 Tyr210Asn 4 Thr217Glu 4 Ala219Gln +

Ser220Glu 4 Gly223Gln Ala204Thr + Tyr213Ala + Pro214Gln 4 Thr215Gln +

Thr217Asp + Ser220Glu Trp20δGln + Tyr210His 4 Thr217Asp 4 Tyr218Met 4

Ser220Asp + Thr224Ser Ala204Asn 4 Gly206Pro + Tyr210Asp 4 Pro214Glu +

Ser220Glu + Thr224Asn Tyr213Ile Ser216Glu + Tyr218His 4 Ala219Glu +

Leu221Asp + Thr224Ser Pro205Ser 4 Trp20δGlu Tyr216Glu Leu221Gly

Ser222Glu + Thr224Gly Ser207Asp 4 Tyr213Met + Pro214Gly + Thr217Gln

Tyr21δAsp 4 Ala219Glu Trp20δPhe 4 Ala219Asp 4 Leu221Ala 4 Ser222Asp 4

Gly223Asn 4 Thr224Glu Gly206Pro + Ile209His + Pro214Glu 4 Ser220Asp 4

Leu221Glu 4 Thr224Pro Trp208Tyr 4 lle209Asn 4 Thr212Asn 4 Thr217Glu 4

Leu221Glu + Ser222Asp ?ro205Gly 4 Ser216Glu 4 Thr217Glu 4 Ala219Gln 4

Leu221Glu 4 Gly223Gln Pro214Asn 4 Thr217Pro + Ala219Asp 4 Ser220Asp 4

Leu221Gln 4 Thr224Asp Ala204His 4 Gly206Asn 4 Ile209His 4 Pro214Glu 4

Ser216Asp + Ser220Glu Ser207Asp 4 Tyr210Glu 4 Thr217Asp + Tyr216Ala 4

Leu221Ile 4 Thr224Pro Thr212Gly 4 Tyr213Ala 4 Pro214Glu 4 Leu221Glu 4

Ser222Asp + Thr224Ser Pro205Gln + Gly206Asn 4 Thr212Gln 4 Tyr216Glu

Ser222Asp + Gly223Asp Pro205Gln 4 Thr212Gly 4 Tyr213Ala 4 Thr217Glu 4

Tyr21δGlu + Ser222Glu Tle209Gln + Tyr213Ala 4 Pro214Glu 4 Ala219Asp 4

Ser222Asp + Thr224Pro Ala204Gly 4 Tyr210Pro 4 Pro214Glu 4 Ala219Glu +

Leu221Asn + Ser222Asp Gly206Pro 4 Thr212Asn 4 Tyr213Thr 4 Pro214Asp 4

Ser220Asp 4 Ser222Glu Ala204Thr + Tyr213Ser Pro214Asp + Ala219Asn 4

Ser220Asp + Ser222Glu Ala204His 4 Gly206Asn 4 Tyr210Pro 4 Pro214Glu 4

Ser220Glu + Ser222Glu Ser207Asp 4 Trp208Met 4 Pro214Glu 4 Thr215Asp 4

Thr217Ser 4 Gly223Pro Trp208Cys 4 Ile209Ser 4 Pro214Glu -*- Ser222Asp +

Gly223Asp + Thr224Gly Pro205Asn 4 Thr212Asn 4 Tyr213Leu + Thr215Asp 4

Ser222Asp + Gly223Glu

TABLE28 Loop 6 - Heptuple Substitution Mutation Variants

Gly206Asn 4 Tyr210Gly Thr212Gln 4 Thr215Gln 4

Tyr21δlle 4 Leu221Ile 4 Thr224Gly Ala204Ser 4 Pro205Ser 4 Ile209Met 4 Tyr213Ser 4

Tyr21δHis 4 Ala219Gln 4 Leu221Asn Ala204Gly 4 Trp208Gln 4 Thr212Gln 4 Ser216Asp 4

Tyr218Gln 4 Leu221Met 4 Gly223Ser Gly206Gln 4 Ile209Leu + Tyr210Ala + Thr212Gly 4

Tyr213Gly + Ser216Asp + Gly223Gln Ala204Gln 4 Pro205Asn 4 Gly206Asn 4 Trp20δlle 4

Tyr210Gln + Thr215Gln 4 Ala219Asp Ala204Asn 4 Pro205Gln 4 Ile209Ala 4 Tyr213Leu 4

Thr217Glu + Tyr21δMet 4 Leu221Gln Pro205Gly 4 Ile209Gln 4 Thr212Ser Ala219Gln 4

Ser220Glu 4 Leu221Cys 4 Thr224Ser Gly206Asn 4 Trp20δCys + Thr212Gly 4 Tyr213Gly 4

Ser216Asp + Thr217Gly + Leu221His Ala204Gln 4 Pro205Ser 4 Tyr210His 4 Thr212Pro 4

Tyr21δPro 4 Ala219Ser 4 Thr224Glu Ala204Gly 4 Gly206Asn 4 Tyr210Cys 4 Tyr213Thr 4

Thr215Gly 4 Giy223Asp 4 Thr224Asn Ala204Gln 4 Pro205Ser + Gly206Gln 4 Thr212A_sn 4

Tyr218Cys 4 Ala219His 4 Gly223Glu Ala204Ser 4 Gly206Asn 4 Ile209Gly 4 Pro214Gly 4

Thr217Gly 4 Leu221Val 4 Thr224Pro Pro205Gly 4 lle209Gln 4 Pro214Asn 4 Tyr218Ile 4

Ala219Gly 4 Leu221Met 4 Ser222Asp Ala204Asn 4 Trp206Ala 4 Ile209Gln 4 Pro214Gly +

Thr217Glu + Tyr216Leu 4 Gly223Asn Gly206Asn 4 Tyr210Glu 4 Tyr213Gly 4 Thr217Gln 4

Tyr213Leu 4 Ala219Thr 4 Thr224Ser Gly20βPro 4 Ile209His 4 Tyr213Asn 4 Pro214Asn 4

Thr215Gln 4 Tyr218Ser 4 Leu221Thr Pro205Gly 4 Gly206Ser 4 Trp206His Tyr213Thr 4

Ala219Gln 4 Ser220Glu 4 Gly223Ser Pro205Gln 4 Gly206Ser 4 Trp206Val 4 Tyr210Leu 4

Thr212Asn 4 Tyr216Met 4 Ser220Glu Ala204His 4 Gly206Asn + Tyr210Ser 4 Pro214Ser +

Thr217Gln 4 Tyr21δHis 4 Ser222Asp Gly206Gln 4 Tyr213Asn 4 Thr215Gly 4 Ser216Glu +

Thr217Gly 4 Tyr21δHis 4 Gly223Asn Gly206Gln 4 Ser207Glu 4 Tyr210Gly 4 Thr212Pro 4

Tyr213Met 4 Gly223Pro 4 Thr224Pro Ala204Asn 4 Trp20δThr 4 Ile209Gln 4 Tyr213Pro 4

Thr217Ser 4 Leu221Pro 4 Ser222Asp Ala204Ser 4 Gly206Gln 4 Trp20δAsn + Thr212Ser 4

Ala219Gln 4 Ser220Glu 4 Leu221Asn Pro205Gly 4 Gly206Gln 4 Ile209Asn 4 Tyr213Val 4

Thr215Gln 4 Thr217Ser 4 Ala219Thr Ala204Ser 4 Pro205Gln + Ile209Asn Thr212Asn +

Thr217Glu + Gly223Pro 4 Thr224Ser Thr212Gly 4 Tyr213Ile 4 Pro214Gly 4 Tyr218Gly 4

Ala219Pro 4 Leu221Asn 4 Thr224Glu Ala204Asn 4 Tyr210Asp + Thr212Pro + Tyr213Asn 4

Thr217Asn 4 Tyr218Cys 4 Leu221His Ala204Gln 4 Ile209Cys + Tyr210Gln 4 Thr212Gly 4

Tyr213Gln Gly223Pro + Thr224Asp Pro205Gln 4 Gly206Gln 4 Trp20δThr 4 Tyr210Leu +

Thr215Gly + Thr217Gly 4 Tyr218Leu Tyr210Ala Tyr213Val + Pro214Gln 4 Ala219Ser

Leu221Cys + Ser222Glu 4 Thr224Asn Ala204Asn 4 Gly206Pro + Ser207Asp + Trp208His +

Ile209His + Pro214Gln 4 Leu221Gln Ala204Asn Gly206Asn 4 Trp208Ile 4 Ile209Asn 4

Ser216Glu + Ala219Pro + Gly223Asn Ala204Ser 4 Gly206Ser + Ile209Ala 4 Thr212Ser +

Pro214Gln 4 Tyr218Asp 4 Leu221His Gly206Asn 4 Ile209Pro 4 Tyr213Cys + Thr215Gln 4 Ser216Glu 4 Thr217Pro 4 Ala219His Ala204Gin - Trp208His + Tyr210Gly + Thr212Gln 4

Tyr218Met 4 Ala219Asn 4 Ser220Glu Ala204Pro + Ser207Asp + Tyr213Val 4 Pro214Gly 4

Thr215Gly 4 Tyr218Ala + Leu221Gln Pro205Asn + Gly206Pro + Trp20δLeu 4 Tyr210Pro 4

Pro214Asn 4 Thr217Gln 4 Tyr21δGlu Pro205Asn 4 Gly206Gln 4 Tyr213Val 4 Ala219Gln 4

Ser220Asp 4 Gly223Ser 4 Thr224Asn Gly206Pro 4 Ile209Cys + Tyr210Ser 4 Thr212Asn 4

Tyr213Cys 4 Pro214Gln 4 Tyr21δCys Ala204Hιs 4 Trp208Asn 4 Thr212Pro + Pro214Gln 4

Thr215Asn 4 Thr217Gly 4 Thr224Asp Pro205Ser 4 Gly206Ser 4 Tyr210Ser 4 Thr215Pro +

Ala219Asn + Leu221Met 4 Ser222Asp Ala204Ser 4 Gly206Gln 4 Trp208Pro + Thr212Asn 4

Thr215Glu + Tyr218Ile 4 Gly223Asn Ala204Ser Pro205Asn 4 Trp20^*8Tyr 4 Thr215Gly 4

Tyr218Pro 4 Ser220Glu 4 Thr224Gly Ala204Asn 4 Tyr210His 4 Pro214Asn 4 Tyr218Pro 4

Ala219Gln 4 Ser220Glu 4 Leu221Ile Ala204Gly 4 Gly206Asn 4 Ser207Asp 4 Thr212Asn 4

Tyr213Leu 4 Thr215Pro 4 Gly223Pro Ala204Asn 4 Tyr210Met 4 Thr212Gln 4 Pro214Gly 4

Ala219Gly 4 Ser222Asp 4 Gly223Gln Tyr210Pro 4 Thr212Gly 4 Tyr213Leu 4 Thr215Gly 4

Thr217Gly 4 Ala219Glu 4 Ser220Asp Gly206Ser + Trp20δAsn Ile209Pro + Ala219Asp 4

Ser220Asp 4 Gly223Ser 4 Thr224Gly Pro205Ser 4 Tyr210Cys + Tyr213Thr 4 Pro214Asp 4

Thr215Asp 4 Tyr21δVal 4 Leu221Ser Trp20δHis + Ile209Val 4 Thr212Pro 4 Tyr213Leu 4

Thr217Ser 4 Ser222Glu 4 Gly223Glu Pro205Gly 4 Gly206Gln Tyr210Asn 4 Tyr213Gly 4

Thr217Gly 4 Ser222Glu 4 Gly223Glu Ala204Thr 4 Thr212Gln 4 Tyr213Gln 4 Thr217Ser 4

Ser222Asp 4 Gly223Asp 4 Thr224Pro Pro205Gly + Ile209Cys 4 Tyr213Val 4 Pro214Gln 4

Thr215Pro 4 Ser222Glu 4 Gly223Asp Trp20δSer Ile209Ser Ser216Glu 4 Thr217Glu 4

Tyr21δGly 4 Ala219His 4 Gly223Pro Gly206Pro + Tyr210Gln 4 Thr212Pro + Tyr213Leu +

Ser216Glu 4 Thr217Asp 4 Leu221Pro Trp206Met 4 He209Gln 4 Tyr210Met 4 Tyr21δGln 4

Ser220Glu 4 Leu221Glu 4 Thr224Ser Ile209Gln 4 Thr212Ser 4 Tyr213Val + Thr215Asp 4

Ser216Glu 4 Tyr21δMet 4 Ala219Pro Tyr213Pro + Pro214Gln 4 Thr215Asp + Ser216Glu +

Thr217Asp 4 Tyr216Met 4 Leu221Ala Ala204His + Pro205Gly + Ser207Glu + Trp208His +

Tyr210His 4 Tyr213Cys 4 Ser222Asp Gly206Pro + Ser207Asp 4 Trp208Ser 4 Pro214Ser 4

Leu221Asn 4 Ser222Asp + Gly223Pro Ser207Glu - Trp208Tyr -^>- lle209Gly 4 Tyr213Thr 4

Pro214Ser Tyr218Met + Ser222Glu Pro205Ser 4 Ser2^~07Asp 4 Trp208Ser 4 Thr212Gly 4

Tyr213Asn 4 Tyr218Gln + Ser222Asp Ala204Thr 4 Gly2^~06Asn + Trp20δThr 4 Thr212Gln +

Pro214Glu 4 Thr215Asp 4 Ser216Asp Ala204Thr 4 Gly206Asn + Ser207Glu 4 Tyr210Ile 4

Leu221Thr 4 Ser222Glu 4 Gly223Glu Ala204Hιs 4 Ser207Glu + Thr215Gln 4 Thr217Gln

Tyr218Asn 4 Ser222Glu 4 Gly223Glu Ala204Gln 4 Trp206Asp 4 Tyr210Thr 4 Thr212Ser 4

Thr217Asn 4 Ser222Asp 4 Gly223Asn Ile209Thr 4 Ser216Asp + Thr217Asp 4 Tyr21δAsp 4

Leu221Ser 4 Gly223Ser 4 Thr224Gly Ala204Asn 4 Gly206Gln 4 Ser207Glu 4 Trp20δHis 4

Thr212Ser + Leu221Asp 4 Ser222Asp Gly206Ser 4 Ser207Asp 4 Tyr210Val 4 Thr212Gln 4

Tyr218Ile 4 Leu221Asp 4 Ser222Asp Ala204Ser + Gly206Pro 4 Pro214Gly 4 Thr215Asp 4

Thr217Asp + Tyr218Pro 4 Gly223Pro Gly206Gln 4 lle209His + Pro214Asp 4 Thr215Pro 4

Ser216Asp 4 Leu221Gly + Thr224Ser Gly206Asn 4 Ser207Glu 4 Trp208Asp 4 Ile209Gly 4

Tyr213Gly 4 Ser220Glu 4 Ser222Glu Pro205Ser 4 Ser207Asp 4 Ile209Asn 4 Tyr213Met 4

Thr217Gln + Ala219His 4 Gly223Glu Pro205Gly 4 Gly206Gln + Ser207Glu 4 Ala219Pro 4

Ser220Asp 4 Leu221Glu 4 Thr224Ser Trp208Met 4 Tyr210Thr 4 Thr212Asn 4 Ala219Gln 4

Leu221Asp 4 Gly223Asp 4 Thr224Gln Pro205Ser 4 Ser207Glu 4 Tyr213His 4 Thr217Pro 4

Ala219Ser + Ser220Glu 4 Ser222Glu Ser207Asp 4 Tyr210Met 4 Pro214Ser 4 Tyr218Asn 4

Ser220Glu 4 Leu221Cys 4 Ser222Glu Ala204Pro 4 Ser207Glu 4 Pro214Gln Ser220Asp 4

Leu221His + Ser222Asp + Thr224Ser Ser207Glu 4 Ile209Met 4 Tyr210Gly 4 Tyr213Leu 4

Thr215Gln 4 Ser220Glu 4 Ser222Asp Gly206Pro 4 Ser207Asp 4 Ile209Ser 4 Tyr210His 4

Thr217Gly 4 Ser222Glu 4 Thr224Asp Pro205Asn 4 Tyr210Cys + Tyr213Gly 4 Thr217Asn 4

Tyr21δMet + Ser220Glu 4 Ser222Asp lle209Gly 4 Thr212Pro 4 Thr215Asn 4 Thr217Gly 4

Ala219His 4 Ser220Glu 4 Ser222Glu Ala204Gln 4 Gly206Gln 4 Thr212Pro 4 Ser220Glu 4

Leu221Val 4 Ser222Asp Thr224Gln Ala204Gln Pro205Ser 4 Gly206Ser 4 Trp20δGln

Thr215Pro 4 Ser220Glu + Ser222Glu Ser207Glu 4 Trp20δLeu 4 Tyr210Ile 4 Thr212Gly 4

Tyr21δCys + Leu221Asp + Gly223Asn Pro205Gly 4 Gly206Pro 4 Ser207Asp Ile209Met +

Leu221Asp 4 Ser222Glu 4 Thr224Asp Pro205Gly 4 Thr215Asn 4 Thr217Pro 4 Tyr21δVal 4 Leu221Thr 4 Ser222Glu + Thr224Asp Trp208Phe 4 Tyr213Met + Thr215Asn 4 Tyr218Asp 4

A_la219Pro 4 Ser220Glu 4 Thr224Gly Ala204Pro -•- Pro205Gly 4 Trp206Leu 4 Thr217Gln 4

Leu221Asp 4 Ser222Glu 4 Thr224Asp Ile209Ala - Thr212Gln -*- Thr217Gln 4 Tyr216Thr 4

5er220Giu 4 Leu221Asp + Gly223Glu Ala204Asn 4 Ile209Cys 4 Tyr213Cys 4 Tyr218Ala 4

Ala219Glu 4 Leu221Asp 4 Ser222Glu Gly206Ser 4 Ser207Glu 4 Trp208His 4 Tyr210Asp 4

Tyr216Val 4 Ala219Thr 4 Ser222Asp Fro205Gly 4 Ser207Asp 4 Trp20δThr 4 Thr212Ser 4

Thr217Gly + Ser220Glu 4 Gly223Ser Ser207Glu + Trp208Asn 4 Tyr210Pro + Pro214Asn 4

Tyr218Ser 4 Ser220Asp 4 Gly223Ser Ala204Gln 4 Ser207Asp + Trp208Gly 4 Ile209Ala 4

Thr212Asn 4 Ser220Glu 4 Gly223Pro Pro205Gln 4 Ser207Asp + Ile209Gln 4 Pro214Asn 4

Thr215Gln 4 Ser220Asp 4 Thr224Gly Pro205Asn 4 Ser207Glu 4 Ile209Val 4 Thr212Gly 4

Tyr213Pro 4 Ala219Asn 4 Thr224Glu Ala204Gly Ser207Glu 4 Ile209Ala 4 Thr212Pro 4

Tyr213Cys 4 Ala219Glu 4 Ser220Asp Pro205Ser 4 Trp20δGlu + Thr212Asn 4 Tyr216Glu +

Ala219His 4 Leu221Glu 4 Ser222Glu Pro205Ser 4 Ile209Pro 4 Tyr210Asp + Thr212Ser 4

Tyr213Ile 4 Ala219Pro 4 Ser222Glu Gly206Ser 4 Trp20δThr 4 Tyr210Ala + Pro214Asp 4

Tyr21δMet 4 Ala219Glu 4 Ser220Asp Ile209Leu 4 Pro214Glu 4 Thr215Pro 4 Ala219Glu 4

Ser220Glu 4 Gly223Pro 4 Thr224Gly Trp20δTyr 4 Thr212Asn 4 Thr217Glu 4 Ala219Gly 4

Ser220Asp 4 Leu221Glu Ser222Asp Ala204Thr 4 Tyr210Pro 4 Tyr213Asn 4 Pro214Glu -

Tyr216Asp 4 Ser220Asp 4 Thr224Gly Gly206Pro Thr212Pro 4 Pro214Gly 4 Thr215Pro 4

Ser220Asp 4 Leu221Asp 4 Thr224Glu Thr212Asn 4 Pro214Gln 4 Tyr21δMet 4 Ala219Asp +

Ser222Glu 4 Gly223Asp 4 Thr224Glu Trp20δGly 4 Tyr210Pro 4 Thr212Gly 4 Ser220Glu +

Leu221Met 4 Gly223Asp Thr224Glu Gly206Gln + Tyr213Asn 4 Pro214Asn 4 Thr217Asp 4

Ala219His 4 Ser220Glu 4 Leu221Asp Pro205Gln 4 He209Pro 4 Tyr210Asn 4 Tyr21δThr 4

Ala219Glu 4 Ser220Asp 4 Gly223Asp Ala204Thr 4 Ser207Asp + Tyr210Asp + Tyr213Met +

Ala219Asn 4 Ser220Glu 4 Thr224Asp Pro205Ser 4 Trp206Pro + Ile209Pro + Tyr213Ile +

Ala219Asp 4 Ser222Asp 4 Gly223Asp Ala204Asn Thr212Pro + Thr217Glu + Tyr216Asp 4

Ser220Glu 4 Ser222Asp + Thr224Gln Gly206Pro 4 Ser207Asp + Trp20δVal 4 Tyr210Thr 4

Tyr213Ala 4 Ser220Asp 4 Thr224Asp 70

?ro205Gly 4 Ser207Glu + Tyr210Ile 4 Pro214Ser 4

Thr217Gly Ser220Asp -^•- Thr224Glu Ala204Thr 4 Tyr210Cys -^ Thr212Pro 4 Tyr218Ile 4

Ser220Glu Leu221Ser Gly223Glu Ala204Gln - Trp208Pro 4 Pro214Asn + Ala219Thr 4

Ser220Glu 4 Gly223Asp + Thr224Gln Pro205Gln 4 Tyr213Gly 4 Thr215Gly 4 Tyr218Ala 4

Ser220Asp 4 Gly223Glu 4 Thr224Pro Pro205Gly 4 Gly206Ser Thr217Pro 4 Ala219His 4

Ser220Asp 4 Leu221Cys + Gly223Asp Pro205Ser 4 Tyr210Thr 4 Thr212Asn 4 Tyr213Cys +

Pro214Ser 4 Ala219Asp 4 Ser222Asp Trp208Asn 4 Thr217Glu + Tyr21δCys 4 Ala219Glu 4

Ser220Asp + Gly223Glu Thr224Ser Pro205Ser + Gly206Pro 4 Trp206Pro 4 lle209A_la 4

Thr215Asp 4 Tyr218Asp 4 Ser220Asp Trp208Thr + Thr212Gly 4 Tyr213Ile + Thr215Asp 4

Leu221Asp 4 Ser222Glu 4 Gly223Glu Ala204Gly 4 Pro205Gly 4 Tyr210Glu 4 Thr217Glu 4

Tyr21δCys 4 A.la219Gly + Leu221Val Ser207Asp 4 Thr215Pro 4 Thr217Asp 4 Tyr21δlle 4

Leu221Ala 4 Ser222Glu 4 Gly223Asp Gly206Pro 4 Trp20δGlu 4 Tyr210Cys 4 Pro214Gln 4

Thr217Asp 4 Ser220Asp 4 Gly223Gln Trp20δThr + Tyr213Pro + Pro214Ser 4 Ser216Asp 4

Tyr218Met 4 Ala219Asp 4 Thr224Pro Ala204Asn 4 Trp208Ala 4 Tyr213Asn 4 Ser216Asp +

Ala219Glu 4 Leu221Cys 4 Thr224Gly Gly206Ser 4 Tyr210Ser 4 Pro214Gly + Ser216Asp 4

Tyr218Cys 4 Ala219Glu 4 Leu221Pro Ala204Thr 4 Tyr210Gln 4 Thr212Gln 4 Tyr213Ala 4

Pro214Gln 4 Thr217Asp + Ser220Glu Pro205Gln 4 Gly206Ser + Ser207Glu 4 Tyr210Ser 4

Ser216Asp 4 Thr217Glu 4 Tyr218Glu Tyr210Met 4 Tyr213Met + Ser216Asp 4 Ala219Glu 4

Ser220Glu 4 Leu221Cys 4 Ser222Glu Ala204Gln 4 Ile209His 4 Ser216Glu 4 Thr217Ser 4

Ala219Glu 4 Ser220Asp 4 Ser222Glu lle209Ser 4 Tyr210Leu 4 Tyr213Leu 4 Pro214Asp +

Thr217Asp 4 Ser220Glu 4 Leu221Glu Ala204His + Pro205Gln 4 Pro214Asn 4 Tyr218Gln 4

Ala219Asp Leu221Glu + Thr224Asp Ala204Gly + Ser207Glu 4 Pro214Glu 4 Tyr218Pro 4

Ser220Glu 4 Ser222Asp 4 Thr224Asn Ala204Pro 4 Pro205Gly Trp20δGlu 4 Ile209Ser +

Tyr210Asp 4 Ala219Thr 4 Thr224Glu Ala204Gly 4 Ser207Glu 4 Trp20δGlu 4 Tyr210Asp 4

Ser216Glu 4 Thr217Gln 4 Thr224Gly Trp206Ala 4 Tyr210Cys + Thr212Ser 4 Thr217Glu 4

Ala219Gln 4 Ser220Asp Ser222Glu Gly206Asn 4 Ser207Glu 4 Trp20δGlu 4 Thr212Ser 4

Ser216Glu 4 Leu221Met 4 Gly223Glu Ala204Thr 4 Ser207Asp 4 Ile209Cys 4 Thr215Asp + _ /. ."I_.e - Ser220Asp + Leu221Asp Ser2^"~A_sp - ThrlllPro 4 Pro214Gln 4 Ser21βG__u +

TyrllSVai - Ser220Asp - Ser222Asp :ie209Pro 4 Thr212Gly - Tyr213Ser 4 Pro214Glu 4

Thr215Glu -*- Ala219Gin Ser220Glu Prc205Asn 4 Ser207Glu 4 lle209Met 4 Pro214Ser 4

Ser216Asp 4 Ala219Glu + Leu221Asp Ala204Thr 4 Ser207Glu 4 Tyr210Leu 4 Ser216Glu 4

Ala219Asp 4 Leu221Asp 4 Gly223Asn Gly206Asn 4 Ser207Glu + Pro214Gly 4 Thr215Glu 4

Thr217Glu 4 Tyr21δThr 4 Ala219Glu Gly206Gln 4 Ser207Glu 4 Tyr210Ala 4 Thr215Asn +

Thr217Asp 4 Ser220Asp 4 Leu221Ala Ser207Glu Trp20δLeu 4 Tyr213Val 4 Pro214Asp 4

Ala219Glu 4 Ser222Glu 4 Thr224Pro Ala204Thr + Ser207Asp 4 Trp20δLeu 4 Tyr210Asp 4

Thr212Pro 4 Thr215Asp -•- Leu221Glu Pro205Gln + Ser207Asp 4 Ile209Ala + Thr217Asn 4

Tyr21δGlu 4 Ala219Asp 4 Gly223Asp Ser207Glu 4 Ile209Thr 4 Tyr213Gly 4 Ser216Asp 4

Ala219Asp 4 Ser222Glu 4 Gly223Pro Gly206Ser 4 Ser207Asp 4 Ile209Ala 4 Thr212Gln 4

Thr217Glu 4 Ala219Glu 4 Leu221Asn Pro205Asn 4 Tyr210Glu 4 Thr217Asp 4 Tyr21δAla +

Leu221Ile 4 Ser222Glu 4 Thr224Pro Ser216Asp 4 Thr217Asp 4 Tyr21δCys 4 Ala219Hιs 4

Leu221Glu 4 Ser222Glu 4 Gly223Asn Ala204Gln 4 Trp206Asp 4 Pro214Gln 4 Ser216Glu 4

Thr217Gln 4 Ser220Asp 4 Gly223Asp Pro205Gln 4 Tyr210Leu 4 Tyr213Thr 4 Ser216Glu 4

Tyr218Glu 4 Ser220Glu 4 Gly223Glu Pro214Gly 4 Thr217Asp 4 Tyr218Gly 4 Ala219Glu 4

Leu221Asn 4 Ser222Glu 4 Thr224Glu Gly206Pro 4 Ser207Asp 4 Trp20δAsp 4 Thr212Gln 4

Thr217Asp + Gly223Ser 4 Thr224Glu Ala204Pro Pro205Ser - Gly206Pro + Ile209Val 4

Tyr213Pro 4 Thr215Asp 4 Ala219Glu Ala204Pro 4 Gly206Pro 4 Tyr210Val + Pro214Glu 4

Ser220Glu 4 Ser222Glu 4 Thr224Pro Ala204Gly 4 Gly206Pro 4 Thr212Gln 4 Pro214Glu 4

Ser220Glu 4 Leu221Val 4 Ser222Asp Ala204Asn 4 Gly206Gln 4 Ile209Ser 4 Tyr210Ala 4

Pro214Glu 4 Ser220Glu 4 Ser222Asp Pro205Gln 4 Trp20δGln 4 Pro214Glu + Thr217Pro 4

Leu221Met 4 Ser222Asp 4 Gly223Glu Ala204Thr 4 Pro205Asn + Pro214Asp Thr215Pro 4

Leu221Cys 4 Ser222Asp 4 Gly223Asp Ile209Pro 4 Tyr210Pro Thr212Pro 4 Thr217Asp 4

Leu221Asn 4 Ser222Asp 4 Gly223Asp Gly20βGln 4 Trp20δThr 4 Ile20^*9Ser 4 Tyr213Met

Thr215Glu 4 Ser222Asp 4 Gly223Glu Pro205Asn 4 Trp20δSer 4 Thr212Gly 4 Tyr213Asn 4

Thr215Asp 4 Ser222Asp 4 Gly223Glu Tyr210Gly 4 Thr212Pro 4 Pro214Gly 4 Ser216Asp 4

Ala219His 4 Leu221Glu 4 Ser222Asp Pro205Gly 4 Gly206Pro 4 lle209His 4 Thr215Asp 4

Ala219Gly 4 Leu221Glu 4 Ser222Glu Pro205Gln + Tyr210Gly 4 Thr212Asn 4 Ser216Asp 4

Ala219Pro 4 Ser220Glu + Leu221Asp Gly206Ser 4 Trp208Glu + Tyr210Gln 4 Thr215Gly 4

Ser216Glu + Thr217Glu + Leu221Pro Pro205Gln 4 Gly206Gln 4 Ser216Glu + Thr217Asp 4 Leu221Ala 4 Ser222Glu 4 Thr224Pro

Gly206Ser 4 Ser207Glu + Trp208Asp + Ile209Leu 4

Thr215Asp 4 Ser216Glu 4 Tyr21δGln Tyr210His 4 Thr212Gln 4 Thr215Asp 4 Ser21βGlu 4

Leu221Glu 4 Gly223Ser 4 Thr224Pro Ala204Ser 4 Pro205Ser 4 Gly206Asn 4 Thr215Asp 4

Ser216Glu 4 Tyr218Val 4 Gly223Asp Ala204Gln 4 Gly206Asn 4 Ser207Glu 4 Trp208Pro 4

Pro214Asp 4 Thr215Asp 4 Ser222Asp Ser207Asp 4 Tyr210Gln 4 Thr212Gln 4 Thr215Asp 4 Ser216Asp + Leu221Gly 4 Ser222Asp

Ile209Met 4 Tyr210Leu 4 Thr217Glu 4 Tyr218Thr 4

Ser220Glu 4 Gly223Glu 4 Thr224Asn Pro205Ser 4 Ser207Glu 4 Trp20δHis 4 Thr215Pro 4

Thr217Asp 4 Leu221Asn 4 Ser222Glu Ala204Thr 4 Ser207Glu 4 Thr212Gly 4 Tyr213Leu 4

Pro214Asn 4 Ser216Glu 4 Ser222Glu Ala204Pro 4 Ser207Asp 4 Tyr210Leu 4 Thr212Gln 4

Tyr213Thr + Thr215Glu 4 Ser222Glu

TABLE 29

Multi-loop Double Mutation Variants

Glyl65Gln 4 Leu221Asp

GlylOδGlu 4 Alal64Gly

Leul04His 4 Thrl67Pro

Alal64His 4 Ser220Glu

Thrl95Gln 4 Ser222Asp

Trp206His 4 Ala219Pro

Gly 69Glu 4 AsnlOβGln

Trpll2Cys 4 Thrll3Pro

Gly 67Gln 4 Trp206Asp

Alall4Glu 4 Leul34Ile

Serl09Glu 4 Glyl36Ser

GlyllOSer 4 Ser207Glu

Vall03Ala 4 Thr212Ser

Alall4Glu + Thrl67Pro

Thrl95Glu 4 Ala204Thr

Gly 67Ser 4 Thr217Glu

Trp20δTyr 4 Ser220Glu

AlalβδHis 4 Thr217Asp

AlalβδGlu 4 Tyr213Asn

GlyllOPro 4 Ser220Asp

Tyrl71Asn 4 Leu221Met

Thr 73Asp 4 Leul34Ser

Thrl37Asn 4 Thr217Pro

Ser 70Asp 4 Alal68Thr

Thrl67Glu 4 Tyr210Pro

Thrl95Glu 4 Tyr210Gly

Tyr210Gln 4 Thr217Glu

Gly 67Glu Valll5Cys

Ser 70Glu 4 Ala219Thr

Asn βδGlu + Tyr210Ser

Glylβ5Asp 4 Prol69Ser

Trp208Pro 4 Thr215Gly

Thr 73Gly 4 Leul04Gly

Thrll3Asn Ser216Glu

Gly 72Gln 4 Glyl39Asp

Tyr210Ile 4 Leu221Gly

Thr 73Pro + Tyrl71Asp

Leul04Gln 4 Thrl37Gly

Gly 69Pro 4 Tyr210Ala

Thr217Gly 4 Gly223Glu

Thrl95Asp 4 Tyr213Ser

Gly 72Asn 4 Ala219Asn

Vall38Pro 4 Tyr210Ala

Tyr218Met + Gly223Asn

Ser220Glu 4 Leu221Asn

Trpll2Ala + Glyl39Asn

Glyl35Glu 4 Leu221Gly

Thrll3Asn 4 Thr217Ser

Thrl95Asn 4 Thr217Asp

Tyr210Val 4 Ser220Glu Vall38Gly 4 Ala219Asp Asnl^ββGlu 4 Leu221Hιs GlylβSGln 4 Leu221Hιs Thrll3Gln 4 Serl92Asp Asn ββAsp Thr 73Ser Thrl67Gln 4 Thr217Gly Valll5Pro 4 Glyl35Ser Ala219Gln 4 Gly223Asp Trp20δHιs 4 Thr217Asn Trp208Leu 4 Tyr210Val GlylOδAsn 4 Serl91Glu Serl07Glu 4 Tyr210Ala Asn βδGln + Ser220Glu Trpll2Cys 4 Leu221Asn GlylOδGln 4 Serl09Asp Vall03Met 4 AlalβδHis Alal64Pro + Serl92Glu Pro214Ser 4 Ser220Glu Thr215Asn Thr217Gln Ser220Glu 4 Leu221Pro Glyl36Ser Trp206Phe Thrl95Asp 4 Tyr210Gln Leul04Gln 4 Ala219Asp Gly 69Pro 4 Leu221Pro Gly 69Asn 4 Ala204Gly Glyl35Glu 4 Ala219Asn Glyl36Asn 4 Ser220Glu Glyl36Ser 4 Trp208Val Glyl39Asp 4 Asnl40Gln Trp208Cys 4 Tyr210Asn Glyl65Ser 4 Ser220Asp GlyllOSer 4 Trp208Thr Alall4Asp Glyl62Asn Serl92Asp 4 Leu221Cys Vall03Ser 4 Glyl35Glu Asnl40Glu + Trp208Ala Thrll3Asp 4 Leu221Ser Serl09Asp 4 GlyllOSer Serl09Glu Thrll3Gln Thr 73Gly 4 Glyl36Asp Alal64His Leu221Asn Gly 67Pro 4 Vall03His Alall4Asn 4 Thr212Gln Asnl4OAsp 4 Gly223Pro Glyl39Gln 4 Pro214Asn Aspl05Glu 4 Thrl95Gly Glyl35Pro 4 Ser207Glu Leul04Glu 4 Alall4Pro Asn ββGlu + Gly 69Pro Valll5Cys 4 Ser207Glu Asn βδGlu + Leu221Ala Asn ββSer 4 Gly 69Asp Serl09Glu 4 Phel93Pro Trp208Glu 4 Thr212Pro

Glyl36Gln 4 Trp206Cys

Ser 70Asp 4 Leu221His

Ser 70Glu 4 Phel93Ser

Thr217Asn 4 Ser222Glu

Tyr213Thr 4 Tyr21δGly

Trp208Ser 4 Leu221Cys

Asnl63Asp 4 Leu221Ser

Asn ββGln 4 Gly223Pro

Glyl39Asn 4 Ser220Asp

Thr 73Ser 4 Pro214Asp

Ser 70Asp 4 Leu221Ile

Glyl65Gln 4 Ser220Asp

AsnlββAsp 4 Thr217Asn

Aspl05Glu 4 ThrlllPro

Asnl4OAsp 4 Trp208Phe

AsnlββGlu 4 Pro214Asn

Trp206Ala + Thr217Ser

GlylOδSer 4 GlyllOGlu

Vall3δAsn 4 Asnl7OAsp

GlyllOGlu 4 Thr224Gln

Trp208Met 4 Ser220Asp

Asn 68Glu 4 Valll5Ser

Asnl70Glu 4 Tyr210Gly

GlyllOAsn 4 Serl92Glu

Trp208Gln Ser220Glu

Asnl40Gln Pro214Gln

Thr217Ser Ser220Glu

Serl41Glu 4 AlalβδThr

Glyl62Glu 4 Tyr210His

Leul04Gly 4 Thr224Asp

Trpll2Pro 4 Serl91Asp

Trp20δThr Tyr210Glu

GlylOδGln 4 ThrlllAsp

Leul34Val 4 Thr217Pro

Asnl OAsp 4 Tyr210His

Valll5Ser 4 Ser220Glu

Gly 69Pro 4 Ser 7OAsp

Thr 73Gly + Tyr210Glu

Gly β7Asp + GlylOδGln

Gly 69Ser GlyllOAsp

Tyr210Thr 4 Ser220Glu

Trp206Phe 4 Leu221Met

Glyl36Gln 4 Leu221Asp

Thrll3Ser 4 Trp208Val

Tyrl71Leu Tyr210Thr

Leul34Asn 4 Thrlβ7Gln

Asnl40Ser 4 Tyr210Asp

Asn ββGln 4 Gly 67Glu

Leul34Cys 4 Pro214Asn

Glyl36Ser 4 Leu221Ala

ThrlllPro 4 Trp208Val

Thrll3Asp + Tyrl71Gly Trp20δlle 4 Ser220Glu Pro214Ser 4 Thr224Glu Glyl65Pro + Ser220Glu Thrl37Gln Tyrl71Glu Trp206Glu 4 Pro214Gly Ser2lβAsp + Leu221Met Glyl36Ser 4 Thr217Gln Serl41Glu 4 Tyr210Leu Trp20δThr + Gly223Ser Asn ββSer + Asnl70Gln Asn ββGln + Asnl70Glu Prol69Glu + Tyr210Thr Tyrl71Leu + Leu221Asp Glyl39Glu + Leu221Pro Thr 73Pro + Leul34Gly

GlylOδSer + Alal64Gln Vall38Ser 4 Trp20δLeu Thr217Ser + Thr224Asp Serl09Asp + Tyr21δAla Asn ββGlu + Phel93Val Asnl4OAsp 4 Ala204Gly Vall38Ser + Ser220Asp GlylOδAsn 4 Tyr210Thr Glyl39Asn 4 Tyrl71Pro Thrll3Pro + Trp20δThr Serl41Glu + Leu22lGly AsnlOβGlu + Thrl67Gly Trpli2Ser + Ala219Ser Aspl05Glu + Asnlβ^βGln Alall4Pro + Tyrl71Asn Thr217Gln + Ser220Asp Thrll3Gly + Tyr210Asp Tyrl71Cys + Tyr210Glu AsnlββAsp + Leu221Val Alall4His + Serl91Asp Gly 67Glu + Thr217Asn Asn ββGlu + Thr215Gln ThrlllAsn + Ser220Glu Trp206Gln + Leu221Ser Gly 69Gln + Alall4Thr Asnl63Asp + Leu221Ala Pro214Gly + Leu221Ile Thr217Gly + Ser220Asp Ala219Thr + Ser220Asp Tyr210Cys + Ser220Glu Vall38Asp + Gly223Pro Thrl95Asn + Leu221Gly Leul04Thr + Thr215Ser Gly 69Gln 4 Leul34Thr

Glyl65Pro 4 Leu221Gly

Thrl67Ser 4 Thr217Glu

Thr212Pro + Thr217Glu

AsnlββSer 4 Tyr218Asp

GlylOδGlu 4 Tyrl71Gln

Gly 67Asn 4 Tyr210Ile

Valll5Cys + Pro214Gly

Thrl37Asp + Tyr210Val

Leul04Met 4 Trp208Gly

Leul34Ala 4 Serl92Asp

Trpll2Gln 4 Ala219Gln

GlylOδGlu 4 Valll5His

Thrll3Asn 4 Glyl39Pro

Trp20δGln 4 Gly223Asn

Ser207Glu 4 Trp206Gln

Asn ββSer 4 Tyr210Asn

Ser207Glu 4 Tyr210Pro

Vall03Ala 4 Thr215Glu

Vall03Asn 4 Tyr210Ala

Asn ββSer 4 Trp20δPhe

Thr215Asn 4 Ala219Thr

Glyl36Asn 4 Glyl39Asn

Tyrl71Thr 4 Ser216Glu

Leul04Glu 4 Tyr210Asn

Serl09Glu 4 Thr217Asn

Leul34Ile 4 AsnlββSer

Tyrl71Gly 4 Thr217Gln

Valll5Cys 4 Thr224Asp

Prol69Ser 4 Thr215Asp

Gly 72Gln 4 Glyl35Asn

Tyr210Thr 4 Ser220Asp

Asn βδGlu 4 Thrll3Ser

AsnlOβGln 4 Thrll3Asp

Serl09Glu 4 Trp20δAsn

Leul34Gln Thrl37Glu

Alall4Thr 4 Asnl70Glu

Ala219Pro 4 Ser220Glu

Tyrl71Val 4 Trp206Pro

Glyl39Ser 4 Tyr213Ser

Serl07Asp Thrll3Asn

Thrl67Gly + Leu221Met

Tyrl71Gly 4 Ser207Glu

Gly 67Ser 4 Thr217Ser

Valll5Gly 4 Thr217Ser

Asn ββSer 4 Trp208Ile

Serl91Glu 4 Trp208Cys

Thrl37Asp 4 Trp20δSer

Leu221Gly 4 Ser222Glu

Ser216Glu 4 Leu221Thr

Glyl62Pro 4 Ser207Glu

Thr217Asp 4 Ala219Thr

Leul04Cys 4 Ser207Glu Gly 69Gln + Ser220Glu Gly 69Gln + Val13δAsp Prol69Gly + Tyr210Leu Thr 73Asp + GlyllOSer Thr215Gly + Thr217Ser Gly β9Ser + Gly 72Asn Serl91Glu + Thr217Asn Glyl35Ser + Tyrl71Ser ThrlllAsn + Phel93Leu Trpll2Leu 4 Ser207Asp Leul04Glu 4 Trp20δAla Leu221Ile 4 Thr224Asp Glyl36Glu 4 Glyl65Asn Tyr210Glu + Thr217Pro Trpll2Phe 4 Pro214Asp GlyllOGln 4 Trpll2Met Asn βδSer 4 Trp208Asp Gly 69Ser 4 Leul04Cys Thrl37Glu 4 Thr215Pro AsnlββGln 4 Thr217Glu Tyr210Thr 4 Thr212Ser Leul34His 4 Trp2OδAsp Serl41Glu 4 Tyr210Val Serl91Glu 4 Tyr210Leu Glyl39Pro 4 Ser207Asp Glylβ2Ser 4 Tyrl71Met Thr 73Gln + Vall03Ala Tyrl71Val 4 Phel93Asp Leu221Thr 4 Ser222Glu Gly 69Asp 4 Alall4Gln GlylOδGlu 4 Trp208Met Gly 72Ser + Tyr210Gln Thrl37Asn 4 Trp2OδAsp Asnl4OAsp 4 Phel93Thr Serl92Glu 4 Trp206Ala Asnl70Glu 4 Pro205Ser Tyr210Ala 4 Tyr213Ser Tyrl71Gln + Tyr218Ile Asn ββAsp + Leu221Cys Asnl63Gln Gly223Asn Vall03Thr Leul34Ser Thrl67Ser 4 Asnl70Asp Ser 70Glu Glyl62Ser Tyrl71Gln + Serl92Glu Tyr210Val + Gly223Ser Thrl37Glu + Leu221Pro Gly 69Asn + Thr217Asp Pro205Ser + Tyr218Glu Tyr210Met + Leu221Asn Thr215Asp 4 Thr217Pro Leul34Val 4 Glyl36Glu Thr 73Pro Leu221Glu Tyr213Val 4 Ser220Glu Thr 73Ser 4 Serl07Glu Trρ20δSer 4 Leu221Pro Thrll3Asp 4 Leu221His Alall4Thr 4 Thr217Asp AlalβδSer 4 Tyr21δGlu Trp208Asp 4 Leu221Asn Thrl37Asp 4 Trp208lle Glyl35Gln 4 Serl91Glu Vall03Glu 4 Trp20δGly Asnl40Asp 4 Leu221His Serl92Asp 4 Tyr210Cys GlylOδAsn 4 Tyr210His Aspl05Glu 4 Trp20δAsn Vall03Pro 4 Tyr210Pro Thr 73Gln 4 Trpll2Tyr Leul34Cys 4 Asnl70Ser AsnlββGlu 4 Tyr210Cys AsnlOβSer 4 Leu221Met Serl09Glu 4 Leu221Cys Serl07Asp 4 Leu221Ala Alall4Ser 4 Leu221Asn Glyl62Asn 4 Leu221Val

TABLE 33

Multi-loop Triple Mutation Variants

Thrl37Asp + Vall3δPro + Tyr210Ala Serl09Glu + Valll5Met + Trp20δCys Glyl39Asn + Ser220Glu + Leu221Asn Trpll2Ala + Glyl35Glu + Leu221Gly Thrll3Asn + Thrl95Asn + Thr2l7Ser A_lal64His + Ser220Asp + Leu221Hιs Asn ββAsp 4 Thr 73Ser + Thrll3Gln

G: 35Ser 4 Thrl67Gln 4 Thr217Gly

Tyrl71Asp Trp20δHιs Thr217Asn GlylOδAsn 4 Trp20δLeu 4 Tyr210Val Trpll2Cys 4 Ser220Glu + Leu221Asn Vall03Met 4 AlalβδHis + Serl91Asp Pro214Ser 4 Thr217Gln + Ser220Glu Thr215Asn 4 Ser220Glu 4 Leu221Pro Glyl36Ser 4 Trp208Phe + Tyr210Gln Gly 69Pro 4 Ala219Asp 4 Leu221Pro Gly β9Asn 4 Ala204Gly 4 Ser220Asp Glyl36Ser 4 Glyl39Asp 4 Trp206Val Asnl40Gln 4 Trp208Cys 4 Tyr210Asn GlyllOSer 4 Glyl65Ser 4 Trp208Thr Vall03Ser + Glyl35Glu 4 Leu221Cys Asnl40Glu + Trp20δAla + Leu221Ser Gly 67Pro 4 Vall03Hιs + Alal64Hιs Asnl4OAsp 4 Thr212Gln 4 Gly223Pro Glyl39Gln 4 Thrl95Gly 4 Pro214Asn Gly 69Pro 4 Leul04Glu 4 Alall4Pro Phel93Pro 4 Trp20δGlu 4 Thr212Pro Glyl36Gln 4 Trp206Cys 4 Leu221Hιs Ser 7OAsp 4 Thr 73Gly 4 Phel93Ser Tyr213Thr 4 Tyr21δGly 4 Gly223Asp Thrl95Glu 4 Trp20δSer 4 Leu221Cys Asnl63Asp + Leu221Ser 4 Gly223Pro Asn ββGln 4 Glyl39Asn 4 Ser220Asp Thr 73Ser 4 Pro214Asp 4 Leu221Ile ThrlllPro 4 Asnl4OAsp 4 Trp208Phe AsnlββGlu Pro214Asn 4 Thr217Ser GlylOδSer 4 GlyllOGlu 4 Trp206Ala Leul34Asp 4 Glyl65Asn 4 Tyr210Thr Phel93Thr 4 Thr217Gln 4 Ser220Asp Gly 67Pro + Trpll2Gln + Serl92Asp Tyr210Ile Thr217Gln 4 Ser222Asp Vall03Ser 4 Leul04Ile + Alal64Asn Alall4Asp Thrl67Gly 4 Thr217Asn ThrlllAsp 4 Thrl95Gly 4 Tyr210Leu Tyr210Thr + Ser220Glu + Leu221Met Glyl36Gln 4 Trp20δPhe + Leu221Asp Thrll3Ser 4 Trp20δVal + Tyr210Thr Leul34Asn 4 Asnl40Ser 4 Thrl67Gln Glyl36Ser + Trp206Val 4 Leu221Ala Glyl65Pro Pro214Ser 4 Ser220Glu Glyl36Ser 4 Tyr210Leu 4 Thr217Gln Serl41Glu + Trp20δThr 4 Gly223Ser Asn ββSer 4 Thrll3Pro 4 Asnl70Glu Thr 73Pro -*- Tyr210Thr + Ser216Glu Tnr 73Pro 4 Tyr210Glu 4 Leu221Ala G:_.139Ser 4 Alal64Ser 4 Tyr210Gly Gl_/108Ser 4 Vall3δSer 4 Alal64Gln Trp203Leu + Thr217Ser 4 Thr224Asp Asn ββGlu 4 Phel93Val + Tyr216Ala GlylOδAsn 4 Tyrl71Pro + Tyr210Thr Thrll3Pro 4 Glyl39Asn 4 Trp206Thr Aspl05Glu 4 AsnlββGln 4 Tyrl71Asn Alall4Pro 4 Thr217Gln 4 Ser220Asp Gly 67Glu 4 Alall4Hιs + Thr217Asn Serl41Glu 4 Trp20δGln 4 Leu221Ser Gly 69Gln 4 Alall4Thr 4 Asnl7OAsp Asnl63Asp 4 Ala219Thr 4 Leu221Ala Ser 70Asp 4 Thr217Gly 4 Ala219Thr Thrl95Asn 4 Leu221Gly 4 Gly223Pro Gly 69Gln 4 Leul04Thr 4 Leul34Thr Glyi39Asp 4 Glyl65Pro 4 Leu221Gly Gly 67Asn 4 GlylOδGlu + Tyr210Ile Vaill5Cys 4 Tyr210Val 4 Pro214Gly Leul04Met 4 Thrl37Asp + Trp20δGly Trpll2Gln 4 Leul34Ala + Serl92Asp GlylOδGlu 4 Valll5Hιs 4 Ala219Gln Thrll3Asn 4 Glyl39Pro 4 Thrl67Asp Thrl37Asp 4 Trp20δGln 4 Gly223Asn Ser207Glu 4 Trp208Gln + Tyr210Asn Asn ββSer 4 Ser207Glu + Tyr210Pro Vall03Asn 4 Tyr210Ala + Thr215Glu Asn ββSer 4 Trp20δPhe Ala219Thr Glyl36Asn 4 Glyl39Asn 4 Thr215Asn Serl09Glu AsnlββSer Thr217Asn Leul34Ile + Tyrl71Gly 4 Thr217Gln Gly 72Gln 4 GlyllOAsp 4 Glyl35Asn Asn βδGlu 4 AsnlOβGln 4 Thrll3Ser Alall4Thr 4 Leul34Gln 4 Thrl37Glu Serl07Asp 4 Glyl39Ser + Tyr213Ser Thrll3Asn 4 Thrl67Gly 4 Leu221Met Gly 67Ser 4 Ser207Glu 4 Thr217Ser Asn ββSer 4 Trp20δlle 4 Thr217Ser Trp20δSer 4 Leu221Gly 4 Ser222Glu Glyl62Pro 4 Ser216Glu + Leu221Thr Leul04Cys 4 AsnlOβGln 4 Ser207Glu Gly 69Gln 4 Prol69Gly + Tyr210Leu Thr 73Asp 4 GlyllOSer 4 Thr217Ser Gly 69Ser 4 Gly 72Asn 4 Thr215Gly Glyl35Ser 4 Tyrl71Ser 4 Serl91Glu ThrlllAsn 4 Phel93Leu 4 Thr217Glu Trp20δAla 4 Leu221Ile + Thr224Asp Trpll2Phe + Pro214Asp 4 Thr217Pro Asn βδSer + GlyllOGln + Trpll2Met Gly 69Ser + Leul04Cys + Trp208Asp Thrl37Glu + AsnlββGln + Thr215Pro GlylOδGlu + Tyr210Thr + Thr212Ser Thr 73Gln 4 Glyl62Ser 4 Tyrl71Met Tyrl71Val 4 Phel93Asp + Leu221Thr Gly 72Ser + GlylOδGlu + Trp20δMet Thrl37Asn 4 Trp208Asp + Tyr210Gln Asnl4OAsp + Phel93Thr + Trp208Ala Gly 69Glu 4 Tyr210Ala + Tyr213Ser Asn ββAsp 4 Tyr218Ile + Leu221Cys Vall03Thr 4 Leul34Ser 4 Asnl63Gln Tyrl71Gln 4 Serl92Glu 4 Gly223Ser Gly 69Asn 4 Thrl37Glu 4 Leu221Pro Pro205Ser 4 Tyr218Glu + Leu221Asn Tyr210Met 4 Thr215Asp 4 Thr217Pro Thr 73Pro 4 Leul34Val 4 Glyl36Glu Thr 73Ser 4 Serl07Glu + Trp208Ser Glylβ2Gln 4 Ser220Asp 4 Leu221Pro Vall03Glu Glyl35Gln 4 Trp208Gly Asnl4OAsp + Tyr210Cys + Leu221His GlylOδAsn 4 Serl92Asp 4 Tyr210His Aspl05Glu 4 Trp20δAsn 4 Tyr210Pro Thr 73Gln 4 Vall03Pro 4 Trpll2Tyr Leul34Cys 4 Asnl70Ser 4 Tyr210Cys AsnlOβSer 4 Serl09Glu 4 Leu221Cys Serl07Asp 4 Alall4Ser 4 Leu221Ala Gly 69Asn 4 Glyl62Asn 4 Leu221Asn Gly 67Ser 4 Glyl35Asp 4 Glyl62Asn Valll5Gly 4 Leul34Ser Alal64Gln Asn ββGlu 4 Leul04Asn 4 AlalβδGly Gly 69Ser 4 AsnlββGlu + Tyr218Asn Th ll3Asp 4 Ala204Ser 4 Tyr218Ser AsnlββGln 4 Trp20δThr 4 Thr217Asp GlylOδGlu 4 Thr217Ser 4 Tyr21δGly AsnlOβAsp 4 Trp20δMet + Thr217Gln Gly 69Glu 4 Tyrl71Val 4 Trp208His Glyl39Asn 4 Prolβ9Gly 4 Trp208Asn Valll5Ala + Thr215Gln + Leu221Glu ThrlllPro 4 Prolβ9Gln 4 Ala219Glu Thr 73Glu 4 Thrl37Asn 4 Trp208His Asn ββAsp + AsnlOβSer + Leu221Thr Gly 67Pro 4 Gly β9Ser 4 Thr224Ser Valll5His 4 Serl91Asp 4 Tyr218Val Thrl37Glu 4 Thrl95Asn 4 Trp208Gly Leul34Glu 4 Trp20δSer 4 Leu221Ser Vall03Ser 4 Trp20δGlu 4 Thr217Asn Glyl39Asn 4 Phel93Pro 4 Leu221Cys AsnlOβGln 4 Alalβ4Pro + AsnlββAsp Aspl05Glu 4 Vall3δSer 4 Thr217Pro Glyl35Pro 4 Tyrl71Thr + Ser222Glu Ser2lβAsp 4 Thr217Asn 4 Thr224Gly Alall4Gly + Asnl40Gln 4 Tyr218Asp Serl07Asp 4 Pro214Gly 4 Leu221Ser Aspl05Glu 4 Asnl70Ser 4 Thrl95Pro Ser 70Glu 4 Tyrl71Leu + Pro214Ser Gly 67Asp 4 Leul04Thr 4 Gly223Asn GlylOδAsp 14Asn + Tyr210Cys GlyllOAsn 14Gly + Ser220Asp Gly 69Asn 69Gln + Ser220Glu Glyl62Glu 08Ala + Thr217Asn Trpll2Tyr 15Pro + Ser220Glu Trp20δGly lOIle + Pro214Asp Leul34Asn 69Ser + Leu221Asp Thrll3Ser 08Leu + Leu221Glu AsnlββSer lδlle + Ser220Glu Trpll2Pro 93His + Pro214Asp Leul04Glu 15Ser + Alal64Ser Gly 69Pro 69Asp + Leu221His GlylOδGln 36Ser + Ser220Glu GlyllOAsn 37Gln + Ser220Glu Alal64Thr ββSer + Tyr210Asp Trpll2Pro 15Pro + Pro214Glu Valll5Glu 39Asn + Tyr210Gln Gly 69Ser 73Ser + Tyr210Pro Alall4Ser 07Asp 4 Thr224Pro Thr 73Pro ββGlu + Thr212Gly Gly 72Gln lOAsp + Leu221His Tyr210Cys 14Asn + Tyr218Asp Gly 69Asn 36Pro + AsnlββSer Thr 73Gly 08Ser + Ser220Glu Thr 73Gly 05Ser + Thr224Ser Vall03Met 64Asp + Leu221Met ThrlllAsn 67Glu 4 Phel93Met Thr 73Asp 10Asn 4 Thr217Gln Asn ββGlu 73Gly + Leu221Gln Thrll3Asp ββSer + Thrl67Pro Asn ββGln 71Gly + Tyr210Pro Asn βδSer 69Pro 4 Asnl70Gln Asn 68Ser 15Glu + Tyr210Asn Thrll3Gln 91Asp + Leu221His Thrl67Pro OδVal + Tyr210Leu Glyl62Glu 08Ala + Thr217Pro Leul04Met 07Asp + Thr212Gly Thrl37Gln 63Asp + Pro205Gln Valll5Thr 17Gln 4 Ser220Asp Ser 7OAsp 3δSer + Tyr210Val Pro214Gly 21Ile Thr224Asp Leul34Val 65Gln 4 Trp20δThr Serl09Asp 14Asn + Tyr210Ser Asn ββSer 15Asn + Trp208Gln Thr 73Pro 93Glu + Gly206Gln Leul34Ser 21Pro + Ser222Asp Valll5Cys 64Glu + Asnl70Gln Alalβ4Asn OδCys + Thr217Glu Asn ββGlu βδPro + Thr215Pro Glyl35Ser 07Asp 4 Thr217Gln Asnl40Gln 67Asp 4 Trp20δMet Glyl65Gln OδThr 4 Leu221Asn Thr 73Pro

OβGlu 4 Leu221Cys Leu221Ile + Gly223Asp Glyl36Gln + Glylβ2Gln Thr217Asn + Thr224Pro Glyl62Glu + Tyr210Gln Asnl63Asp 4 Thr2l7Gly Thr217Asn 4 Leu221Glu Tyr210Met + Thr217Glu Serl91Glu 4 Trp206Cys Glyl39Asn 4 Pro205Asn Trp208Ala 4 Tyr210Pro Thrl95Glu 4 Tyr210His Trp208Met 4 Tyr210Pro Alal64Gly 4 Tyr210Glu Pro205Gln 4 Leu221Asp Serl41Asp 4 Leu221Asn Thrl67Gln 4 Thr217Pro Asnl70Glu 4 Leu221Pro Leul34Ser 4 Thr224Asn Ser207Asp 4 Tyr21δThr Alal64Asn 4 Ala203Ser Trp208Glu + Thr212Ser Trp20δAsn 4 Tyr210Thr Serl92Glu + Thr217Ser Serl09Asp + Tyr210Gln Asnl63Gln 4 Phel93Pro Prol69Glu + Leu221Ser Ser220Glu 4 Leu221Met Tyr210Asp 4 Thr217Pro Tyr210Ser Leu221Pro Serl92Glu 4 Leu221Ala Thrlβ7Ser 4 Thr217Glu Glylβ2Pro + Tyr210Ala Glyl62Asn 4 Serl91Glu Tyr21δPro 4 Leu221Glu Alal64Gln Pro214Ser Trp206Tyr 4 Thr217Asn Thr212Ser 4 Ser220Glu Glyl39Gln 4 AlalβδAsp Pro214Gln 4 Leu221Gln Trp20δTyr 4 Thr215Gln Glyl62Asn 4 Phel93Ile Thr217Asn 4 Ser220Asp Glyl39Glu 4 Thr212Asn Tyr213Ala 4 Thr217Pro Thr217Glu 4 Gly223Pro Thr217Asp 4 Leu221Ala Thr217Asp 4 Tyr218Asn Vall03Ala 4 Ser220Glu Tyr210Gly + Giy223Ser Tyr210Asn + Thr217Asp Glyl39Gln 4 Thr217Gly Leul04Ile Tyr210Ile

Prol69Asn 4 Thr217Ser Tyr210Gly 4 Pro214Gly + Leu221Glu

Leul34Ile -*- Ala219Glu + Ser220Asp

Asnl OAsp 4 Serl41Glu + Tyr216Ser

Tyr213VaI 4 Ser220Glu 4 Leu221Glu

Tyr210Val 4 Ser220Glu + Leu221Asp

Alal64His 4 Ser220Glu 4 Leu221Glu

Tyrl71His 4 Ser220Asp + Leu221Glu

Tyr210Ile 4 Ser220Glu 4 Leu221Asp

Trpll2Leu 4 Ser220Asp 4 Leu221Glu

Glyl36Pro 4 Serl91Asp 4 Serl92Glu

AlalβδSer 4 Thr217Asp 4 Tyr21δGlu

AsnlββSer 4 Thr217Glu 4 Tyr21δAsp

Glyl62Asp 4 Asnl63Glu 4 Leu221Met

Trp208Ser 4 Tyr210Glu 4 Ser220Asp

Alall4Thr 4 Glyl65Asp 4 Phel93Glu

Ser207Glu 4 Thr217Asn 4 Ser222Glu

Asnl40Ser 4 Trp206Glu 4 Ser220Asp

Trp208Asp 4 Leu221Asn 4 Ser222Glu

Glyl39Gln 4 Trp208Glu 4 Leu221Asp

Asn βδGlu 4 Aspl05Glu 4 AsnlOβAsp

Thrl37Gln 4 Glyl39Asp 4 Tyrl71Glu

Trp208Asp 4 Tyr210Glu 4 Leu221Ala

Gly 69Pro 4 AsnlββGlu 4 AlalβδGlu

Tyrl71Cys 4 Tyr210Glu 4 Leu221Asp

Thrl37Glu 4 Prol69Asp 4 Asnl70Ser

Glyl62Glu 4 Tyrl71Asp 4 Thr217Ser

Leul34Ile 4 Serl91Glu 4 Gly223Glu

Valll5Pro 4 Ser207Glu 4 Ser220Asp

Glyl39Pro 4 Ser207Asp 4 Ser220Glu

Ser 70Glu 4 Vall03Pro 4 AsnlOβAsp

Gly 69Asp 4 Thr 73Asp 4 Tyr216Ile

Asn ββAsp 4 Trp208Gly 4 Pro214Glu

Asn ββAsp 4 Ser 70Glu 4 Glyl36Ser

Asnl63Asp 4 Serl91Asp 4 Ser207Glu

Asn ββSer 4 Gly 69Asp 4 Serl09Glu

Gly 67Glu 4 Trp20δlle 4 Thr215Asp

Ser 70Glu 4 Tyr210Gln 4 Thr217Glu

Ser 70Glu 4 Glyl35Pro + Thr217Glu

Vall38Glu 4 Serl41Asp 4 Tyr210Gln

Vall38Glu + Serl41Glu + Pro214Asn

Serl07Asp 4 GlyllOAsp 4 Alall4Glu

Serl09Glu 4 Alall4Glu 4 Glyl36Ser

GlyllOGlu 4 Glyl3βGlu 4 Tyr210Gln

Glyl35Ser 4 Tyr210Asp 4 Thr217Glu

Ser 70Glu 4 Prol69Asn 4 Leu221Asp

Valll5Gly 4 Glyl62Asp 4 Thrl67Asp

GlyllOPro 4 Thr217Asp + Ser220Asp

Alal64His 4 Thr217Asp + Ser220Asp

Tyr210Cys 4 Thr217Asp 4 Ser220Asp

Vall03Ser 4 Thr217Asp Ser220Asp

Thr217Glu 4 Tyr216Val 4 Ser220Glu

Gly 69Ser 4 Thr217Glu 4 Ser220Glu

Tyr210Val 4 Thr217Asp 4 Ser220Glu Gly 67Glu + Serl07Glu 4 ThrlllAsn AsnlββAsp + Serl91Asp + Leu221Val Leul34Asp + Phel93Glu 4 Gly223Asp Asn ββAsp 4 Ser2lβAsp + Thr217Glu Vall03Glu + AsnlOβGlu 4 Thrll3Glu Ser 70Asp 4 Glyl39Ser 4 Ser220Asp Leul04Asp 4 Aspl05Glu 4 Serl91Glu Alall4Glu Ser2lβAsp 4 Thr217Glu Thrl95Glu 4 Ser2lβAsp 4 Thr217Glu Valll5Glu 4 Ser22OAsp 4 Leu221Glu Serl09Asp 4 Ser22OAsp 4 Leu221Glu Thr215Glu Ser2lβAsp 4 Ser220Asp Serl92Asp 4 Phel93Glu 4 Thr217Asp Glyl65Asp 4 Tyr2lOGlu 4 Ser220Asp Ser 70Glu 4 Aspl05Glu Tyr218Asp Glv 69Glu 4 AlalβδGlu Thrl95Glu

TABLE 34

Multi-loop Quadruple Mutation Variants

A.sn ββGln 4 Leul04Gly 4 Tyr210Pro 4 Thr217Glu Gly 67Ser 4 Gly 69Ser 4 Vall03Gln 4 Gly223Asp Trpll2Cys 4 Alal64Glu Trp20δGln 4 Thr217Gly Leul34Ile + Trp20δGln 4 Ala219Gly + Ser220Glu Leul04Ala + Serl09Glu 4 ThrlllGln 4 Valll5Ala GlylOδGlu + Alal64Gly + Glyl65Gln 4 Thrl67Pro Gly 69Glu + AsnlOβGln 4 Trpll2Cys + Thrll3Pro Thr 73Asp 4 Leul34Ser 4 Thrl37Asn 4 Thr217Pro Gly 67Ser 4 Glyl36Pro Thrl95Ser 4 Leu221Ala Thr 73Gly 4 Leul04Gly 4 Trp208Pro Thr215Gly GlyllOGlu 4 Glyl36Pro 4 Phel93Met 4 Thr217Pro Alal64Gln 4 Tyrl71Ile 4 Phel93Ile 4 Thr224Gly Glyl36Pro + Alal64Thr 4 Ser207Asp 4 Trp20δLeu GlylOδGln + Trpll2Pro 4 Trp208Thr 4 Tyr210Glu ThrlllAsp + Leul34Val 4 Tyr210His 4 Thr217Pro Glyl36Gln Trp208Phe 4 Ala219Asp + Leu221Met Thrll3Ser Tyrl71Leu + Trp208Val + Tyr210Thr Glyl36Ser Serl41Glu + Trp208Thr + Tyr210Leu Asn ββSer + Thr 73Pro 4 Thrll3Pro + Asnl70Glu Thr 73Pro + Glyl39Ser Tyr210Glu Leu221Ala Glyl08Ser Vall38Ser 4 Trp208Leu + Thr217Ser Asn 68Ser 4 GlyllOGln 4 Trpll2Met 4 Serl92Asp Thr 73Gln 4 Vall03Ala 4 Glyl62Ser 4 Alal64Glu Thrl37Asn + Asnl4OAsp 4 Phel93Thr 4 Trp206Ala Leul34Ser 4 Asnl63Gln 4 Ser220Glu + Gly223Asn Asnl40Glu + Tyrl71Gln 4 Tyr210Val + Gly223Ser Thr 73Pro + Leul34Val + Glyl3βGlu 4 Thr217Pro Serl07Glu + Glyl62Gln + Trp206Ser 4 Leu221Pro Glyl08Asn 4 Glyl62Asn 4 Serl92Asp 4 Tyr210Cys Vall03Pro + Serl09Glu 4 Trp208Asn Tyr210Pro Thr 73Gln 4 Trpll2Tyr 4 Leul34Cys 4 Asnl70Ser Gly 69Asn 4 Glyl62Asn 4 AsnlββSer 4 Leu221Asn Gly 67Ser 4 Leul34Ser 4 Glyl62Asn Alal64Gln

Leui34Ile 4 Glyl35Pro 4 Alal^βδThr 4 Trp206Met

Trpll2Ser 4 AlalβδHis 4 Thr217Ser 4 Leu221Ala

GlyllOSer 4 ThrlllGln 4 Thr215Ser 4 Ser222Asp

ThrlllPro 4 Thrl37Asn 4 Prol69Gln 4 Ala219Glu

Thr 73Glu 4 AsnlOβSer 4 Trp206His 4 Leu221Thr

A_sn ββAsp 4 Gly 67Pro 4 Gly 69Ser 4 Thr224Ser

Leul04Cys 4 Valll5His 4 Serl91Asp 4 Tyr21δVal

Leul34Glu 4 Thrl95Asn 4 Trp208Ser 4 Leu221Ser

Vall03Ser 4 Glyl39Asn Thr217Asn 4 Leu221Cys

AsnlOβGln 4 Alal64Pro 4 Asnl^ββAsp 4 Phel93Pro

Aspl05Glu 4 Glyl35Pro 4 Vall38Ser 4 Thr217Pro

Gly 67Asp 4 Thrll3Asn 4 Alall4Asn 4 Tyr210Cys

AsnlββAsp 4 Thrl95Gly 4 Tyr210Gly + Thr224Gln

Trpll2Pro 4 AsnlββSer 4 Phel93His + Tyr218Ile

Gly 69Pro + Leul04Glu 4 Valll5Ser + Alal64Ser

Trpll2Pro 4 Valll5Glu 4 Glyl39Asn 4 Tyr210Gln

Gly 69Ser 4 Thr 73Ser 4 Tyr210Pro 4 Thr224Pro

Gly 72Gln 4 Thr 73Pro 4 GlyllOAsp 4 Leu221His

ThrlllAsn 4 Glyl36Pro 4 Tyr210Thr 4 Tyr218Asp

Glyl08Asp 4 Glyl36Asn 4 AsnlββSer 4 Leu221Thr

Vall03Met 4 Pro205Ser 4 Leu221Met 4 Thr224Ser

Gly 69Asn 4 Thr 73Asp 4 Trp20δGln 4 Tyr210Leu

Thr 73Gly 4 AsnlββSer 4 Thrlβ7Pro 4 Leu221Gln

Asn ββGln 4 Thrll3Asp 4 Tyrl71Gly 4 Tyr210Pro

Glyl62Glu 4 Thrl67Pro 4 Trp208Val 4 Tyr210Leu

Ser207Asp 4 Trp208Ala 4 Thr212Gly 4 Thr217Pro

Leul04Met 4 Thrl37Gln 4 Asnl63Asp 4 Pro205Gln

Serl09Asp 4 Leul34Val 4 Glyl65Gln 4 Trp208Thr

Alall4Asn 4 Valll5Asn 4 Trp20δGln 4 Tyr210Ser

Asn ββGlu 4 Alal64Asn 4 Trp20δCys + Thr215Pro

Glyl35Ser 4 AlalβδPro 4 Ser207Asp + Thr217Gln

Thrl^β7Asp 4 Trp208Met 4 Thr217Gln Leu221Asn

Thr 73Pro 4 AsnlOβGlu 4 Thrll3Ser + Leu221Cys

Glyl62Gln 4 Serl91Glu 4 Thr217Asn 4 Thr224Pro

Gly 69Gln 4 Leul04Gln 4 Glyl62Glu 4 Tyr210Gln

Leul04Gln 4 Tyrl71Pro 4 Thr217Gly 4 Leu221Glu

GlyllOGlu 4 Phel93Asn 4 Trp208Cys 4 Gly223Pro

Alal64Gly 4 Serl91Glu 4 Pro205Asn 4 Trp206Cys

Glyl35Pro 4 Ser207Glu 4 Trp206Ala 4 Tyr210Cys

Thr 73Glu 4 Leul04Val 4 Pro205Gln 4 Leu221Cys

Trpll2Cys 4 Vall38Thr 4 Thrl67Gln 4 Thr217Pro

Serl09Asp 4 Alalβ4His 4 Gly223Pro 4 Thr224Asn

Asn βδGln 4 Tyr210Pro 4 Thr212Ser 4 Ser220Asp

Leul34Gly 4 Trp206Glu 4 Tyr210Met + Thr212Ser

Asnl63Asp 4 Asnl70Gln 4 Trp208Asn 4 Tyr210Thr

Trpll2Gly Trp20δSer 4 Tyr210Asp 4 Thr217Pro

Trpll2Met 4 Serl92Glu 4 Trp208Ser 4 Tyr210Val

Trpll2Leu 4 Ser220Asp 4 Leu221Ala 4 Thr224Asn

Leul04Met 4 ThrlllAsp 4 Alal64Gln 4 Pro214Ser

Glyl39Gln 4 Glyl65Pro 4 Thr212Ser 4 Ser220Glu

Thrll3Asp 4 Leul34His 4 Trp206lle 4 Leu221Ala

Aspl05Glu Pro214Gln 4 Thr215Gln 4 Leu221Gln Leul34Pro 4 Glyl62Asn 4 Gly2O^βSer Trp20δTyr Trpl12 la + Phel93Ile 4 Thr217Asn Ser220Asp Serl07Giu -^L Thr212Asn 4 Tyr213Ala Thr217Pro ly c7G1n Gly 69Gln + Thrl13Asn Ser220Glu βόGln 4 Trp208Cys 4 Tyr2lOAsn Thr217Asp

Thr 73Giu + Leul04Ile 4 Glyl39Gln Thr217Gly

Thrl13Ser 4 Prol69Asn 4 Tyr2lOIle Thr217Ser

Leul34Pro Thrl67Gln 4 Tyr2lOMet Ser220Asp

Thr 73Giu + AsnlββGln 4 Tyrl71His Leu221Ala

Vail3δMet 4 Asnl70Gln 4 Thr217Asn Ala219His

Glyl36Gln 4 Ala219Ser 4 Ser22OAsp Thr224Gln

Gly 69Asn AsnlββGln 4 Tyr2lOGlu Thr217Gly

Thr 73Ser 4 Glyl65Gln 4 Thr212Pro Ser222Glu

Thrl37Pro 4 Asnl40Gln 4 Tyrl71Gly Ser220Glu

Tyr2lOPro 4 Tyr213Ile 4 Ser2 2OAsp Leu221Pro

Vail03Asp AsnlOβSer 4 Phel93His Thr217Gly

Asn βδAsp 4 Thrll3Asn 4 Vail 38Ala Trp20δHis

Aspl05Glu + Leul34Met 4 Glyl65Ser Leu221Gln

Asn 68Gln 4 Trp208Thr 4 Thr217Ser Ser220Asp

Tyrl71Asn 4 Thrl95Asn 4 Tyr210Asn Tyr21δAsn

Asn ββSer 4 Prol69Ser 4 Tyr2lOCys Gly223Glu

Alal14Gln 4 Phel93Glu 4 Tyr2lOGly Thr224Pro

Asn ββSer Glyl36Ser 4 Alal64Asp AlalβδGln

Leul04Gly Glyl62Pro 4 Serl92Asp Leu221Asn

Glyl08Asp Glyl62Pro 4 Trp208Thr Gly223Asn

Asn ββGlu Thrll3Gly -*- Alal64Pro Gly223Pro

GlyllOPro 4 Trpll2Ala 4 Ser2lβGlu Leu221His

Vail15Met 4 Prol69Ser 4 Trp2OδHis Tyr210Asp

Leul04Ser 4 Serl09Asp 4 GlyllOPro Thrl95Gly

Leul04Asn 4 Thrl37Glu 4 Trp208Ser Leu221Val

AsnlOβGln 4 Alall4Asp 4 Tyrl71Thr Leu221Pro

Pro205Asn 4 Tyr210Asn 4 Thr217Gly Tyr216Leu

Leul04Pro 4 AsnlOβSer 4 Thrl37Asp Thr217Pro

GlyllOSer Thrl37Gln 4 Asnl70Glu Th 217Asn

Thrl13Gly 4 Phel93Met 4 Trp208His Tyr213Ile

AsnlββGln Ser207Asp 4 Thr217Ser Tyr218Met

Glyl62Asp 4 Phel93Pro 4 Trp2OδTyr Thr217Gln

Gly 69Ser 4 Serl07Glu 4 Trp2OδMet Tyr210Leu

Vail03Asp 4 Thrl67Pro 4 Ile209Val Tyr210Gln

ThrlllAsn 4 Asnl70Ser 4 Trp2OδTyr Thr212Gly

Serl07Glu + Thrl37Pro 4 Glyl62Ser Trp208Cys

Vail03Thr 4 Alal64Thr 4 Alal^βδGlu Thr217Ser

Glyl39Pro 4 Tyr210Asn 4 Leu221Pro Thr224Pro

Leul34Ser 4 Serl41Glu + Tyr210Ala Thr217Gln

Vail03Asn 4 Leul34Ala 4 Tyr218Met Thr224Asn

Asn ββAsp + Glyl35Gln + Asnl63Ser Leu221Val

Gly 69Glu 4 Valll5Pro 4 Thrl37Pro Trp20δlle

Gly 69Pro GlylβSPro 4 Tyr2lOSer Leu221Ile

Asn ββGln 4 GlyllOGlu 4 Trpl12Gly Trp206Pro

Trpl12Ala + Alall4Pro 4 Glyl35Ser Asnl63Asp

Gly 67Asn 4 Ser207Glu 4 Tyr2lOThr Thr217Pro

AsnlββAsp 4 Thrl67Pro 4 Ile209His Tyr210Val

Thr 73Ser 4 AsnlOβGln Tyrl71Glu Thr217Asn Valll5Glu - Tyr210Ser 4 Thr217Gln 4 Ala219Gly

Leul04Pro + Thrl95Gln 4 Trp208Tyr 4 Ser222Asp

Serl41Asp 4 Ala204Gln Trp208Val 4 Tyr210Leu

Gly 67Ser 4 Gly 69Glu + Vall03Gly 4 Tyr210Gln

Tyrl71Met 4 Phel93Leu 4 Thr215Asp 4 Thr217Gln

Asn ^βδGln 4 Aspl05Glu 4 Trp20δPhe 4 Leu221A_la

Leul04Asn 4 Valll5Gln + Leul34Gln 4 Pro214Glu

Glyl65Ser 4 Serl91Asp 4 Pro214Gly 4 Leu221Thr

Gly 67Ser 4 Asn βδGln Tyr210Val 4 Ser220Asp

Serl09Asp 4 Vall36Gly 4 Trp206Ser 4 Thr217Pro

ThrlllGly 4 Glyl36Pro 4 Serl41Asp 4 Leu221Ile

Trpll2Asn 4 Glyl62Ser 4 Ile209Cys 4 Thr224Gln

Valll5Ala 4 Prol69Glu 4 Tyr210Val 4 Leu221Cys

Glyl35Ser 4 Tyr210Leu + Thr217Glu 4 Leu221Pro

Asn ββGln 4 Serl09Asp + GlyllOGln 4 Tyr218Thr

Thr 73Gly 4 Trp206Ile Thr217Glu 4 Leu221Asn

Leul34Ser Trp208His 4 Tyr210Leu 4 Ser220Glu

Giyl35Pro 4 Thrl37Gln 4 Ala219Ser 4 Leu221Gln

Asn 68Gln 4 AsnlOβGlu 4 Prol69Gln 4 Thr215Gln

Vall38Gln 4 Glyl65Glu 4 Tyr21δLeu 4 Leu221Cys

Phel93Gln 4 Ser207Asp 4 Trp20δCys 4 Thr217Gln

Gly 67Asn 4 Glyl65Asn 4 Phel93Tyr 4 Pro205Ser

Asn ββSer 4 AlalβδSer 4 Tyr210Cys 4 Thr217Asp

Pro214Gly 4 Ser216Asp Thr217Gln 4 Leu221His

Asn ββGln 4 Valll5Gln + Asnlβ^βAsp 4 Tyr218Met

ThrlllGlu 4 Glyl65Ser + Ala219His + Leu221Thr

Serl41Asp 4 Asnl63Ser 4 Phel93Tyr 4 Ala204Ser

GlylOδPro 4 Asnl63Ser 4 Glyl65Asp 4 Tyr213Ser

Trp20δHis 4 Tyr210Thr 4 Tyr21δGln 4 Ser220Asp

Gly 69Glu 4 Alal64Pro 4 Trp206Val 4 Thr224Ser

Serl09Asp 4 Ala203Pro 4 Trp20δHis 4 Thr217Ser

Thr 73Asp 4 Tyrl71Cys 4 Tyr210Pro 4 Leu221Pro

Glyl62Pro 4 Tyr218Val + Ala219Thr 4 Ser220Glu

Asnl70Gln 4 Serl92Glu 4 Trp208Pro 4 Tyr210Gly

Thrll3Ser 4 Thrl37Gln 4 Phel93Ser 4 Pro205Ser

Trpll2Pro 4 Thrl37Gly 4 Ser220Asp 4 Leu221Asn

Glyl62Glu 4 Alalβ4Ser 4 Alal68His 4 Pro214Asn

Alall4Pro 4 Leul34Glu 4 Asnl40Ser 4 Phel93Ile

Alal64Gln 4 AlalβδGlu 4 Trp208Asn 4 Tyr210Leu

Vall03Pro 4 Glyl39Pro 4 Asnl63Asp 4 Glyl65Gln

Glyl62Ser 4 Trp208Glu 4 Tyr210Ser 4 Leu221Gln

Serl41Asp 4 Tyrl71His 4 Trp208Ala 4 Leu221Met

Asn ββSer 4 Glyl39Gln 4 Asnlβ3Gln 4 Tyr210Thr

Leul34Asp 4 Glyl36Ser 4 Asnl63Gln 4 Trp208Val

Serl91Asp 4 Phel93Leu + Pro214Gln 4 Thr217Ser

Gly 67Asn 4 GlylOδAsp + Thrl95Asn + Thr215Pro

Alall4Thr 4 Asnl40Glu + AsnlββSer 4 Ala203Pro

Serl91Glu 4 Ala203Gly + Trp208Asn 4 Tyr210Ala

Asnl4OAsp 4 Serl41Giu 4 Alal64Gin 4 Tyr216Ser

Thr 73Ser 4 Tyr213Val 4 Ser220Glu 4 Leu221Glu

Phel93Met 4 Thr215Gln 4 Ser220Glu 4 Leu221Glu

Thrll3Ser 4 Trp206Leu 4 Ser220Glu 4 Leu221Glu

Thr 73Pro 4 Valll5Ala + Serl92Asp 4 Phel93Glu A_iall4Asn 4 Trp20δPro 4 Tyr210Glu 4 Ser220Glu Trp208Cys + Tyr210Glu 4 Ala219Asn 4 Ser220Asp Alall4Pro + Glyl39Pro 4 Tyr210Asp 4 Ser220Glu Tyr210Glu + Thr217Gln 4 Tyr216Gln 4 Ser220Glu Asnl40Ser + Trp206Glu 4 Ser220Asp 4 Leu221Asp Vall03Pro 4 Trp208Glu 4 Tyr210Ser 4 Ser220Glu Thr 73Gln 4 Serl07Asp 4 Serl09Glu 4 Asnl63Gln Gly 67Gln 4 Serl07Asp + Serl09Glu 4 Leu221Cys A.splOSGlu 4 AsnlOβSer + Serl09Glu 4 Thr217Pro Leul04Glu 4 Aspl05Glu 4 Serl07Asp 4 Leu221Ser Gly206Pro 4 Pro214Asp 4 Thr217Glu 4 Leu221Asn Ser207Asp 4 Trp208Glu 4 Ser220Asp 4 Leu221Ser Glyl35Asp + Glyl62Glu 4 Tyrl71Asp + Thr217Ser Gly 69Glu 4 Leul04Asp 4 Aspl05Glu 4 Tyr210Ala AlalβδAsn 4 Ser220Asp Leu221Pro 4 Ser222Asp Phel93Gln 4 Ser207Glu 4 Thr217Ser 4 Leu221Glu GlyllOGln 4 Glyl65Gln 4 Ser222Asp 4 Thr224Asp AsnlOβGlu 4 Serl09Asp 4 Phel93Ile 4 Trp208Leu GlyllOAsn 4 Thrl37Glu 4 Prol69Asp 4 Asnl70Ser Glyl36Glu 4 Glylβ2Glu 4 Trp206Ala 4 Thr217Asn Leul34Ile 4 Serl91Glu 4 Leu221His 4 Gly223Glu Glyl39Pro 4 Tyrl71Met 4 Ser207Asp 4 Ser220Glu Vall03Pro 4 ThrlllGly 4 Ser207Asp 4 Ser220Asp GlylOδAsn 4 Thrl95Gly 4 Ser207Asp 4 Ser220Asp Ser 70Glu Vall03Pro 4 AsnlOβAsp 4 AlalβδPro Thrll3Gly 4 Glyl62Ser 4 Asnl63Asp 4 Asnl70Glu Leul04Ile 4 Serl07Glu 4 GlyllOAsp 4 Asnl70Gln Thr 73Gly 4 Glyl35Asp 4 Prol69Asp 4 Trp20δThr Tyr210Met 4 Thr215Asp 4 Tyr21δGlu + Leu221Asn Leul04Thr 4 Glyl3βGlu 4 Prol69Asp + Leu221Val Asn ββAsp 4 Thr 73Glu 4 Tyr216Ile 4 Leu221Cys Aspl05Glu 4 ThrlllAsp 4 Alall4Pro 4 Vall38Met Asn βδGlu 4 Thr 73Glu 4 Trp208Tyr 4 Thr217Asn Ser207Glu + Tyr213Ile 4 Ala219Asp + Ser222Asp Asnl63Asp + Alal64Ser 4 Serl91Asp Ser207Glu Thr 73Glu + AlalβδSer 4 Tyr210Glu Tyr218Glu Gly 67Glu + Trp208Ile + Pro214Asn + Thr215Asp Aspl05Glu + ThrlllGlu 4 Thrll3Glu 4 Asnl63Gln Ser 70Glu 4 Tyr210His 4 Thr217Asp 4 Leu221Ala Gly 67Glu 4 GlylOδAsp 4 Thrl37Gln 4 Thrlβ7Pro Serl07Asp 4 GlyllOAsp 4 Alall4Glu 4 Tyr216Asn Ser 70Asp 4 Tyr210Glu 4 Thr217Asn 4 Ser220Glu Thrll3Gly 4 Trp208Glu 4 Ser220Asp + Thr224Asp Vall3δMet 4 A-la219Glu 4 Ser222Asp 4 Thr224Gln Ser 70Glu 4 Trpll2Tyr 4 Tyr218Cys + Leu221Glu Gly 67Ser 4 Ser 70Glu 4 Prol69Asn + Leu221Asp Valll5Cys Glyl3 Asn 4 Thr217Asp Ser220Asp Gly 69Ser 4 Valll5Thr 4 Thr217Glu 4 Ser220Glu AlalβδHis 4 Trp208Tyr 4 Thr217Asp 4 Ser220Glu ThrlllGln 4 Glyl35Pro 4 Thr217Asp 4 Ser220Glu Alal64Thr 4 Serl91Glu + Trp208Glu Thr215Asn Asn βδGlu 4 Vall03Glu 4 Serl09Glu 4 AsnlββGln Ser 70Asp + Glyl39Ser 4 Thr217Glu 4 Ser220Asp Leul34Asp 4 Phel93Glu 4 Ala203Ser 4 Gly223Asp Asn ββAsp 4 Asn βδSer 4 Ser216Asp 4 Thr217Glu Alal64Giu 4 Trp20δCys 4 Leu221Glu 4 Ser222Asp Ser 70Glu 4 Ser207Glu 4 Ala219Ser 4 Ser220Glu Gly 67Asp 4 Ser 70Asp 4 Thr 73Gly 4 Tyr210Glu Trp20δlle Thr217Glu 4 Tyr218Asp 4 Ser222Asp Leul04Ile 4 ThrlllGlu 4 Glyl35Asp 4 Asnl63Gln Alall4Glu 4 Leul34Ile 4 Ala219Glu 4 Ser220Asp AsnlββGiu 4 Thrl67Gly 4 Ser222Asp 4 Gly223Asp Glyl39Glu 4 Tyr210Ala 4 Ser222Asp 4 Gly223Glu Vall03Gly 4 GlyllOGlu 4 ThrlllGlu 4 Ser222Glu Alall4Glu 4 Asnl70Ser 4 Ser2lβAsp 4 Thr217Glu GlylOδAsp 4 Serl09Asp 4 Tyr210Gln 4 Thr217Glu Asn βδSer 4 Serl09Asp 4 GlyllOGlu 4 Ser220Glu Asn βδGlu 4 Asnlβ3Ser 4 Ser207Asp 4 Trp206Glu Ser207Asp 4 Trp20δGlu 4 Thr217Glu 4 Leu221Cys Gly 69Pro 4 Aspl05Glu 4 AsnlOβAsp 4 AsnlββAsp Serl09Asp 4 Tyr210Gln 4 Ser220Glu 4 Leu221Asp Tyrl71Gly + Serl92Glu 4 Ser220Asp 4 Leu221Glu Asnl70Glu 4 Serl91Glu 4 Serl92Glu 4 Pro205Ser Asn ββAsp 4 Tyr210Gln 4 Thr217Glu Tyr218Glu AsnlOβAsp 4 Serl07Glu 4 Thrl37Asp 4 Tyr210Gln Serl92Asp 4 Phel93Glu Pro205Ser 4 Thr217Asp Ser 70Asp 4 Glyl62Asp 4 Asnl63Glu 4 Leu221Ser Gly 69Asn 4 AsnlOβGlu 4 Tyr210Asp 4 Ser220Glu Vall03Ser 4 Asnl4OAsp 4 Tyr210Glu 4 Ser220Asp Gly 69Glu 4 Phel93His 4 Ser207Giu 4 Ser222Asp Glyl62Glu 4 Asnl63Gln 4 Trp208Asp 4 Ser220Glu Valll5Cys 4 Asnl7OAsp 4 Trp20δAsp 4 Ser220Asp GlyllOGlu 4 Trp20δAsp 4 Leu221Asn 4 Ser222Glu Prol69Asp 4 Tyrl71Glu 4 Pro214Asn 4 Gly223Glu Asnl63Glu 4 Ser207Glu 4 Trp208Met 4 Tyr210Thr Gly β7Glu 4 Ser 70Glu 4 Valll5Cys 4 Glyl62Glu Glyl62Glu 4 Alal64Glu 4 Ser220Glu 4 Leu221Gly Asn ββGlu 4 Vall03Asp 4 GlylOδAsn 4 Leu221Met Aspl05Glu 4 Serl09Glu 4 Tyr21δAsp 4 Leu221Ala Gly 67Asp 4 Alal64Asp 4 AlalβδGlu 4 Tyr213Asn Gly 69Pro 4 Serl41Asp 4 Pro214Asp 4 Thr217Asp Thr 73Glu 4 Trpll2Ile 4 Trp206Gly 4 Ser220Asp Gly 72Ser 4 Serl07Asp 4 ThrlllAsp 4 AsnlββGln Glyl65Glu 4 Serl92Asp 4 Thr217Asp 4 Leu221Ile Glyl35Glu 4 Asnl40Glu 4 Ala204His 4 Pro214Ser Aspl05Glu 4 GlyllOAsp 4 Asnl7OAsp 4 Ala219Gly Asn ββAsp 4 Asn 6δAsp 4 Leul34Gly 4 Asnl63Glu Leul04Thr 4 AlalβδAsp 4 Asnl7OAsp 4 Ser220Glu Thr 73Asp 4 Serl09Glu 4 Pro214Glu 4 Thr217Asn Thr 73Asp 4 Valll5Cys 4 Pro214Asp 4 Thr224Glu Serl09Asp 4 Thrll3Asp 4 Leul34Asn 4 Phel93Pro Gly 69Ser 4 GlylOδAsp 4 GlyllOAsp 4 Ser220Glu Glyl39Asp 4 Serl41Asp 4 Alal64Asp 4 Leu221Val Asn ββAsp 4 Trp208Met 4 Ser220Asp 4 Ser222Glu Trpll2Asp 4 Tyr210Thr 4 Ser220Glu 4 Ser222Glu Serl09Asp 4 Ser207Glu 4 Tyr210Gly 4 Leu221Glu Gly β9Asp 4 AsplOSGlu 4 Thrl37Asp 4 Trp20δAsn Serl09Asp 4 ThrlllGlu Serl91Glu 4 Tyr210Leu Glyl39Gln -*- Prol69Asp + Serl91Asp 4 Ser207Glu Giy 67Ser 4 Asnl4OAsp + Ser222Asp 4 Thr224Glu VaIl38Asp + Asnl70Glu + Trp208His 4 Thr215Glu Asn βδAsp 4 ThrlllGly + Vall36Asp 4 Asnl7OAsp Leul04Glu 4 Serl07Asp + Glyl35Gln 4 Tyrl71Asp Leul04Pro + Trpll2Asp + Asnl40Glu + Ser222Glu Glyl39Glu 4 Asnl7OAsp 4 Gly206Pro 4 Ser220Glu Alal64Asp 4 Asnl7OAsp + Ser2lβAsp + Thr217Pro Thrll3Glu 4 Alall4His 4 Ser207Glu + Ser220Glu GlyllOGlu 4 Valll5Gln 4 Ser207Asp + Thr224Glu AsnlOβGlu 4 Asnl63Glu 4 Asnl70Glu + Leu221His Gly 69Asp 4 Thr 73Glu 4 Thrll3Asn + Serl41Glu Gly 69Asp 4 Thr 73Asp 4 ThrlllGly 4 Glyl39Glu Serl07Asp 4 Asnl63Glu 4 Serl92Asp 4 Trp20δMet Asn βδAsp 4 GlylOδAsp 4 Ser207Asp 4 Ile209Ala Glyl35Asp 4 Glyl39Pro 4 Prol69Asp 4 Trp20δGlu Asn ββGlu 4 Thrl67Gly 4 Trp20δGlu 4 Pro214Asp Asn ββAsp 4 Serl09Asp 4 Trp20δGly 4 Pro214Glu Gly 69Glu 4 Asnl40Ser 4 Serl92Glu 4 Pro214Asp Leul04Gly 4 Glyl39Glu + Serl91Asp 4 Thr224Asp AsnlOβGln 4 Thrll3Asp 4 Asnl63Asp 4 AsnlββGlu Leul34Glu 4 Ser207Asp 4 Trp20δPro 4 Tyr210Glu Serl91Glu 4 Thrl95Asn 4 Tyr21δPro 4 Leu221Glu Asnl7OAsp + Phel93Ser 4 Ser220Glu 4 Thr224Asp Aspl05Glu 4 Alall4Glu 4 Serl92Glu 4 Thr224Asn Gly 67Asp 4 GlylOδAsp 4 Ser207Asp 4 Tyr210Cys Asn βδAsp + GlyllOGlu 4 Thrll3Gly 4 Leu221Ala Leul04Ile 4 Alall4Glu 4 Ser220Glu 4 Gly223Glu Serl07Asp + Ser220Glu + Leu221Pro Gly223Asp Vall03Asp 4 Vall3δGlu 4 Serl41Glu 4 Gly206Gln Vall38Glu 4 Serl41Asp 4 Tyr210Gln 4 Ser220Asp Asn βδAsp 4 Valll5Gly 4 Thr217Asp 4 Ser220Glu Asn ββAsp 4 Leul04Gly 4 AlalβδThr Ser216Glu Vall03Glu 4 AsnlOβGlu 4 Thrll3Pro 4 Ser220Asp Aspl05Glu 4 Thrll3Asp 4 Glyl39Asn 4 Serl41Glu Ser 70Glu Aspl05Glu 4 Glyl39Asp Tyr210Ala Asn ββGlu 4 Tyr210Asp 4 Thr217Glu + Leu221Ala GlylOδGlu 4 Glyl35Ser 4 Tyr210Asp 4 Thr217Glu Gly 69Asp 4 GlylOδGln 4 Trpll2Asp Serl41Glu Serl07Glu GlyllOPro 4 Thr217Asp 4 Ser220Asp Glyl35Glu + Thr217Glu + Tyr218Val 4 Ser220Glu Asnl63Asp 4 Tyrl71Thr 4 Thr217Asp 4 Ser220Asp Alall4Thr 4 Thrl37Glu + Thr217Asp 4 Ser220Asp AsnlββGlu 4 Thr212Pro 4 Thr217Asp + Ser220Asp Gly 67Asp 4 Serl07Asp + Tyr210Asn 4 Ser222Glu Asnl40Ser Trp20δGly 4 Thr217Glu + Leu221Asp AsnlββGln + Thr215Pro + Thr217Glu Leu221Asp Valll5Gln 4 Alal68Gln + Phel93Asp Leu221Asp Trp208Ile + Pro214Ser 4 Ser220Glu 4 Thr224Glu GlylOδGlu 4 Thrl37Glu 4 Asnl63Asp 4 Trp20δSer Glyl39Pro 4 Alalβ4Glu 4 Serl91Asp 4 Pro214Glu Thr 73Asp 4 ThrlllPro 4 AsnlββAsp 4 Serl91Asp Ser 70Gl + Alal^βδHis + Thrl95Glu 4 Ser216Asp Ξerl09Giu 4 Thrl37Asp 4 Glyl39Ser 4 Alal64Gln Asni70Gin 4 Tyr210Asp 4 Pro214Glu 4 Gly223Ser Ser 70Glu 4 Glyl39Asn 4 Alal64Glu 4 Ser220Asp Asn ββAsp + Thrll3Glu 4 Serl41Asp 4 Trp208Gly Asn ββAsp 4 GlylOδGlu 4 Thr215Gly 4 Leu221Ser Asn βδSer 4 Thr 73Glu AsnlββGlu 4 Leu221Asp Thr 73Asp 4 Serl92Asp 4 Ser220Asp 4 Leu221Met Serl07Asp 4 ThrlllAsp 4 Serl92Glu 4 Thr217Ser

TABLE 35

Multi-loop Quintuple Mutation Variants

Asn ββGln + Gly 67Ser 4 Leul04Gly 4 Tyr210Pro 4

Thr217Glu Gly 69Ser 4 Vall03Gln 4 Trpll2Cys 4 Thr217Gly 4

Gly223Asp Vaill5Pro + Leul34Ile 4 Alal64Glu 4 Phel93Asn 4

Trp20δGln Leul04Gly + Thrll3Ser 4 AlalβδGlu 4 Asnl70Ser 4

Ala219Gly Gly 69Ser 4 Serl09Asp + ThrlllGln 4 Valll5Ala 4

Tyr210Pro Vall03Thr 4 GlylOδGlu 4 Valll5Pro 4 Tyrl71Ser 4

Trp206Asn Leul24Ala 4 Vall3δCys 4 Asnl63Gln 4 Tyrl71Ser 4

Ser220Asp Asn ββSer + Valll5Gly 4 Serl91Glu 4 Trp208Ile 4

Thr217Ser Gly 72Asn 4 Trpll2Phe 4 Valll5Ala + AsnlββGln 4

Trp208Gly Leul34His 4 Glyl65Gln Trp20δPro 4 Tyr210Val 4

Thr217Glu Leul04Pro + Tyrl71Leu 4 Trp20δLeu 4 Ala219Glu 4

Leu221Ser AsnlββGlu + Thrl67Pro 4 Tyr210Pro 4 Tyr213Ile 4

Pro214Asn Glyl39Gln 4 Asnl70Glu 4 Trp206Cys 4 Thr215Ser +

Leu221Asn Alall4Glu + Glyl35Ser 4 AlalβδThr 4 Ala203Thr 4

Trp208Met Alal64His 4 AlalβδHis 4 Phel93Ala 4 Thr217Ser 4

Ser220Asp Gly 67Pro + Gly 69Ser 4 Serl91Asp 4 Tyr216Val +

Thr224Ser Glyl39Asn 4 Alal64Pro Phel93Pro 4 Thr217Asn 4

Leu221Cys Leul04Met + Ser207Asp 4 Trp20δAla 4 Thr212Gly +

Thr217Pro Leul34Val + Trp208Thr 4 Pro214Gly 4 Leu221Ile 4

Thr224Asp Serl09Asp 4 Alall4Asn 4 Valll5Asn 4 Glyl65Gln 4

Tyr210Ser Leul04Ser GlyllOPro 4 Glyl3βGlu 4 Alal68Thr 4

Ala219Gln GlyllOPro ÷ Leul34Pro 4 AsnlββAsp 4 Asnl70Ser 4

Trp208Asn Thril3Gln 4 Prol69Gly 4 Asnl70Gln 4 Tyr218Gln 4

Leu221Ser Thrl37Ser + Thrl95Gln 4 Ser207Glu + Trp20δGln 4

Thr217Pro Gly 69Gln + Vall03Gly 4 Trpll2Met 4 Trp20δAsn 4

Pro214Gly Glyl36Pro Glyl62Ser 4 Trp208Ile + Tyr210Gly 4

Pro214Gln Thrl37Asp + Glyl65Gln 4 Thrl95Ser 4 Trp20δPro 4

Thr212Ser Asn ββSer 4 Prol69Ser 4 Tyr210Asn 4 Tyr21δAsn 4

Gly223Glu Ser 70Glu 4 AsnlOβGln + Vall36Ala 4 AlalβδGln 4

Leu221Val Gly 69Ser 4 Thrll3Asp 4 Alall4Gln 4 Trp20δThr 4

Ala219Ser Thrll3Gln 4 AlalβδAsp + Prolβ9Ser 4 Asnl70Ser 4

Ala219Thr Glyl39Ser 4 Asnl70Gln 4 Tyrl71Leu 4 Trp20δCys 4

Thr215Ser Gly 67Ser 4 Alall4Asn 4 Glyl39Ser 4 Tyr210Thr 4

Tyr21δSer Trpll2Glu + Alal64Pro 4 Tyrl71Pro 4 Thrl95Asn 4

Thr217Ser Gly β7Glu Valll5Asn 4 Glyl62Gln 4 Glyl65Gln 4

Phel93Val Glyl35Ser 4 Tyrl71Cys + Trp206His 4 Tyr210Met 4

Leu221Val Gly 69Asn AsnlOβSer 4 AsnlββGlu 4 Tyr210Cys 4

Leu221Gly Glyl35Ser + Tyrl71Asn 4 Trp20δHis + Ser216Glu 4

Leu221Gly Gly 69Asn + Serl09Asp 4 Glyl65Asn 4 Phel93Asn 4

Trp20δHis Prol69Ser 4 Ser207Glu + Pro214Asn 4 Thr215Asn 4

Thr217Gly Ser 70Asp Asnl63Ser 4 Tyr210Val + Tyr213Asn 4

Leu221Ala GlylOδGln GlyllOSer 4 Glyl35Gln 4 Thrl37Asn 4

Trp206Glu Asn βδSer 4 Alal64Gln 4 Asnl70Glu 4 Thr217Ser 4

Gly223Ser Gly 69Ser 4 Trpll2Cys 4 Alal64Gly 4 Leu221Ser 4

Gly223Glu Gly 69Glu 4 Gly 72Gln -<- Thrl37Pro 4 Trp206Ile 4

Tyr21δPro Vall03Gln Vall36His 4 Glyl62Ser 4 AsnlββAsp Trp208His Valll5Gln Leul34Cys 4 AlalβδSer 4 Prol69Asp 4

Tyr210Ser Gly 67Gln 4 Trpll2Leu 4 Phel93His 4 Thrl95Glu 4

Thr217Ser Gly 67Gln 4 Leul04Asn 4 Alal64Glu 4 Ala204Ser 4

Pro205Ser Glyl35Ser + Vall36Ser 4 Asnl40Glu 4 Tyr210Leu 4

Thr217Gly Gly 67Asn 4 Thr 73Ser 4 Tyrl71Leu 4 Phel93Tyr 4

Tyr210Pro Glyl35Pro 4 Trp206His 4 Tyr210Leu 4 Ala219Ser 4

Leu221Gln Asn βδGln 4 AsnlOβGlu 4 Thrl37Gln 4 Prol69Gln 4

Thr215Gln Thrl37Asp Vall36Gln 4 Trp20δlle 4 Tyr21δLeu 4

Leu221Cys Gly 67Asn 4 Glyl65Asn 4 Phel93Tyr 4 Pro205Ser 4

Trp206Cys Asn ββSer 4 Thr 73Ser 4 Serl07Asp + AlalβδSer 4

Tyr210Cys Thrl37Gly 4 Tyr210Pro 4 Thr215Pro 4 Ser216Asp 4

Thr217Ser Thr 73Pro 4 Trpll2Asp 4 Glyl39Pro 4 Thr215Pro 4

Leu221His Leul04Cys 4 ThrlllPro 4 Trp208Pro 4 Leu221Cys 4

Ser222Asp Thrll3Gln 4 Ala204His 4 Trp206Phe 4 Thr217Asp 4

Leu221Cys ThrlllGly 4 Glyl39Gln 4 Tyrl71Leu 4 Tyr210Met 4

Thr217Glu GlyllOAsn + Valll5Gln + Prolβ9Gln 4 Thrl95Gly +

Ser220Asp Tyrl71Asp 4 Gly206Ser 4 Thr217Gln 4 Ala219Gly 4

Leu221Thr Serl09Glu 4 ThrlllSer 4 Valll5Cys 4 Vall3δCys 4

Tyrl71Ser Leul^'04His 4 Asnl63Asp 4 Trp208Thr 4 Tyr210Cys 4

Thr217Asn Glyl36Asn 4 Glyl39Pro Serl41Glu 4 Prolβ9Gln 4

Ala219His Gly 69Ser 4 Glyl36Ser 4 Thrl37Asn 4 Thrl95Pro 4

Ala219Glu AsnlOβAsp Glylβ2Ser 4 Asnl63Gln 4 Prol69Gly 4

Leu221Pro AlalβδGly + Phel93Met 4 Pro205Asn 4 Tyr210Asn 4

Leu221Asn Gly 69Pro 4 Glyl39Asp 4 Alal64Gly 4 Tyr213Ser 4

Thr217Ser Gly 72Ser Leul04Gln 4 AsnlββGlu Prol69Asn 4

Thr215Gly Leul34Cys 4 Trp20δAsn 4 Tyr210Ala 4 Tyr216Gly +

Thr224Gly Leul04Gln 4 Thrl95Asn 4 Trp208Val 4 Tyr210Pro 4

Leu221Cys Gly δ7Pro 4 Thr 73Asn 4 Trp20δPro 4 Ala219Asn 4

Ser220Asp Thr 73Gln 4 GlylOδPro 4 Glyl39Pro 4 Serl92Glu 4

Gly223Gln Asn 68Glu + Thr 73Gln 4 Prol69Gln 4 Tyr210Pro 4

Leu221Met Leul04His 4 Thrll3Gln 4 Asnl40Gln 4 Thrl67Asn 4

Phel93Asp Gly 67Pro 4 Asn 68Ser + Glyl39Asp Thr215Gly 4

Thr217Pro Glyl36Ser 4 Vall38Met 4 Thrl67Gln 4 Tyr21δAsn 4

Ser220Asp Aspl05Glu 4 GlyllOSer Trpll2His 4 Tyr210Gln

Ala219Thr Asn βδAsp AsnlOβGln 4 Alal64Ser Thrl95Gln 4

Trp20δCys Leul04Val 4 Leul34Ala 4 Thrl37Asn 4 Asnl70Asp 4

Leu221Ile Gly 67Ser 4 Leul34Met 4 Trp208Ala + Tyr210Ser 4

Thr217Pro Alall4Gly 4 Glyl35Asp 4 Glyl39Ser 4 Asnl70Ser 4

Thr217Ser ThrlllAsn 4 Phel93Ser 4 Pro205Gln 4 Trp208Thr 4

Leu221Glu Serl09Asp 4 Vall36Asn 4 Phel93Ala + Trp20δCys 4

Thr212Gln Ser 70Asp 4 AsnlββSer Ile209Pro 4 Tyr210Met 4

Leu221Ser AsnlOβSer 4 Leul34Cys 4 Glyl36Ser 4 AlalβδThr 4

Tyr210Glu Gly 67Gln 4 Leul34Gln 4 Glyl35Asp + Phel93His 4

Thr224Gly Asn βδSer 4 Leul04Ala 4 Glyl62Asp Tyrl71Met

Thr217Gly Asnl63Glu + Thr212Gln 4 Thr215Asn Thr217Asn +

Leu221Met Gly β9Gln + AsnlOβGln 4 Ser207Glu + Trp20δThr 4

Thr217Gly Glyl39Glu + AlalβδHis 4 Thrl95Gly + Leu221His 4

Thr224Asn Gly 72Ser 4 Alall4Asn 4 Leul34His + Prol69Glu 4

Trp20δTyr Vall36Ser 4 Alal64Asn 4 Tyr210Cys + Tyr218Leu +

Ser220Asp Leul04Gln 4 Tyrl71Glu 4 Trp208Gly Thr217Gln 4

Ala219His AsnlOβSer 4 Tyrl71Gly 4 Phel93Gln + Thr215Gln 4

Ala219Gln Gly 72Ser 4 GlylOδAsn 4 Trpll2Ile + AsnlββGlu +

Leu221Cys Asn βδSer Leul04Thr 4 Thr215Ser + Ala219Thr 4 Thr224Asp Vall38Ser Asnl63Asp 4 Phel93Cys 4 Pro205Asn 4

Thr217Ser Gly 67Ser 4 Tyr210Pro 4 Tyr218Thr 4 Ser220Asp 4

Leu221Ser Glyl35Asn 4 Glyl36Gln 4 Glyl62Ser 4 Tyr210Asn 4

Pro214Asn Thrl95Ser 4 Trp208Pro 4 Tyr210Gly 4 Ala219Ser 4

Leu221Ile Asn βδGlu 4 ThrlllPro 4 Asnl63Ser 4 Phel93Tyr 4

Leu221Val Gly 67Ser 4 Thr 73Pro + Asnl70Glu 4 Tyr210Leu 4

Leu221Val Glyl35Asn 4 Glyl39Ser 4 Phel93Asp 4 Thr217Gly 4

Leu221Gly Glyl36Gln + Glyl39Gln 4 Glyl65Ser 4 Tyr210Asp +

Thr217Pro Serl41Glu 4 Glyl62Pro 4 Thrl67Gly 4 Tyr210Ile 4

Thr217Ser Leul04Pro 4 GlyllOPro 4 Glyl36Pro 4 Pro205Asn 4

Thr217Glu Valll5Ala 4 Alal64Thr 4 Serl92Asp 4 Trp20δLeu 4

Tyr210Met Thr 73Asn 4 Vall03Asn 4 Thr215Asn 4 Thr217Gln 4

Ser222Asp Vall03Pro 4 Trp206Tyr 4 Ile209Cys 4 Tyr210Thr 4

Thr217Gln Trpll2Phe 4 Alall4Ser 4 Prol69Gln 4 Ser220Glu 4

Leu221His GlylOδAsn 4 Leul34Met 4 Tyr210Ser 4 Tyr213Ile 4

Pro214Glu AlalβδPro 4 Trp20δHis 4 Tyr210Ala 4 Ser220Glu +

Leu221Gln Glyl39Pro 4 Asnl40Glu 4 Serl41Asp 4 AsnlββSer 4

Trp20δThr Vall03Asn 4 ThrlllGly 4 Tyr210Gln 4 Ser216Asp

Thr217Glu Vall3δHis 4 Glyl39Asn 4 Ser207Asp 4 Trp20δGlu +

Leu221Gln AlalβδAsn 4 Prol69Gly 4 Thr217Pro 4 Ser220Asp +

Leu221Asp Tyr210Leu 4 Ala219Gly 4 Ser220Asp 4 Leu221Asp 4

Gly223Ser Vall03Ala 4 Prol69Asn 4 Thr217Gln 4 Ser220Asρ +

Leu221Asp Thr 73Asn 4 Asnl70Asp 4 Tyrl71Glu + Tyr210Ile +

Leu221Ser Glyl36Ser 4 Serl91Asp 4 Serl92Glu 4 Tyr216Cys +

Leu221Asn Thr 73Pro 4 Valll5Ala 4 Serl92Asp 4 Phel93Glu +

Trp208Gln Thrl37Gln 4 Glyl62Asp 4 Asnl63Asp + Ala203His 4

Pro205Gln Asnl63Gln 4 Tyr210Asp + Thr215Pro + Ser220Glu 4

Gly223Pro .AsplOSGlu 4 AsnlOβGlu + Serl07Asp + Trp20δPhe +

Tyr210Ser Trpll2Gly 4 Phel93Gln 4 Ser207Asp 4 Trp20δHis 4

Ser222Asp GlylOδAsn 4 Ser207Glu + Tyr213Thr + Thr215Pro +

Ser222Asp Leul34Met + Ser207Asp 4 Trp20δVal + Tyr210His 4

Ser222Asp Gly 69Glu 4 AsnlOβAsp 4 Alall4His 4 AlalβδSer 4

Thr217Gln Leul04Pro 4 Trp206Glu 4 Thr212Asn + Thr215Ser 4

Ser222Asp ThrlllPro + Thrl37Asn 4 Prol69Gln 4 Tyr210Glu +

Ala219Glu Thr 73Gly 4 Valll5Gly + Trp206Glu + Tyr210Asp 4

Ser220Glu Glyl35Gln 4 Glyl36Glu 4 Asnl70Ser 4 Tyrl71Glu

Trp20δGln Thr 73Ser 4 Serl07Asp 4 Serl09Asp 4 Glyl36Pro +

Tyr210Gly Leul34Pro 4 Prolβ9Gln 4 Pro214Glu + Ser21βGlu +

Thr217Gly Aspl05Glu 4 Serl09Glu 4 Prol69Gly 4 Tyrl71Gln 4

Thr212Ser Glyl35Pro 4 Vall36Ala 4 Ser207Asp 4 Tyr210Thr 4

Gly223Glu Glyl35Pro 4 Ser207Glu 4 Trp206Ala 4 Tyr210Pro

Gly223Glu Prol69Gln + Phel93Val 4 Tyr210Glu 4 Thr215Asn 4

Leu221Asp Alall4Gln 4 Glyl36Ser Ala219Asn 4 Ser220Asp 4

Ser222Asp Leul34Ser 4 Thrl67Gly 4 Ser220Asp 4 Leu221Pro 4

Ser222Asp GlylOδGln 4 Trpll2Leu 4 Tyr216Glu 4 Ser220Asp 4

Leu221Glu GlylOδPro 4 Alall4Asn 4 Ser207Asp 4 Tyr210Glu 4

Ser220Glu Thrl37Glu 4 Glyl62Glu 4 Prol69Asn 4 Asnl70Ser 4

Tyrl71Asp Asn βδGln + Glyl62Glu 4 Alal64Thr Glyl65Asp 4

Tyrl71Ala Thrl37Pro Glyl39Asp 4 Asnl70Glu 4 Trp206Met

Tyr210Thr Asn βδGln 4 Vall3δSer 4 Glylβ2Pro 4 Alal64Glu 4

Asnl7OAsp Asn βδGln 4 Ser207Glu 4 Trp20δThr 4 Thr217Ser +

Ser220Asp Vall38Ala 4 Phel93Thr 4 Ser207Asp 4 Tyr210Met +

Ser220Asp Phel93Ala Trp208Glu 4 Ile209Ser 4 Leu221Cys + Gly223Glu TnrlllGlv ^■*- Phel93Gln + Ser207Asp 4 Ser220Asp 4

Gly223Glu Ser 70Asp 4 AsplOSGlu + GlylOδGlu 4 ThrlllSer

Tyr210Val Glyl36Glu 4 Glyl62Asp 4 Glyl65Ser 4 AlalβδPro 4

Thrl95Glu Leul04Gly 4 Glyl65Glu 4 Prol69Glu 4 Thr217Asn 4

Leu221Ala Thrl95Asn Tyr210Asp 4 Thr217Glu 4 Ser220Glu 4

Gly223Pro Trp208Phe 4 Tyr210Asp 4 Tyr213Cys 4 Thr217Glu 4

Ser220Glu Ser 70Asp 4 GlylOδGlu 4 Tyrl71Gly 4 Thr217Gln

Leu221Pro Thrl95Gly 4 Ser207Glu 4 Tyr210Glu 4 Tyr213Cys 4

Ala219Ser Asn βδGlu 4 Aspl05Glu 4 GlyllOAsp + ThrlllPro 4

Asnl40Ser Ser 70Asp 4 Vall03Gln 4 Pro214Gln 4 Ser220Asp 4

Leu221Glu Asn ββAsp 4 Thr 73Glu 4 AsnlOβSer 4 Trp20δHis 4

Leu221Thr Alal64Asp 4 Thrl67Asp 4 Tyrl71Pro 4 Ala219Gln 4

Leu221Thr Alall4His 4 Ala219Gly 4 Ser220Glu 4 Ser222Glu 4

Thr224Glu AsnlOβAsp + Serl09Asp 4 ThrlllAsp 4 Thrl37Ser 4

Pro205Asn Leul04Cys + Asnlβ3Glu + Serl92Asp 4 Ala219Pro 4

Ser222Glu Glyl62Asp 4 Alal64Gln 4 Ser207Asp 4 Trp206Tyr 4

Gly223Asp Ser 70Asp 4 Serl07Glu + Serl09Glu 4 Valll5Cys 4

Glyl35Ser Alall4Gly 4 Ala219Asp 4 Ser220Glu 4 Leu221Asn 4

Gly223Glu Asn ββAsp 4 Aspl05Glu 4 Valll5Gln 4 Thrl37Ser 4

Trp20δHis Gly 69Glu + Thr 73Glu 4 Vall03His Serl07Asp +

Leu221Ala GlylOδPro + Trpll2Gln 4 Glyl62Asn 4 Ser220Glu +

Gly223Asp Asn βδAsp 4 Asnl70Gln 4 Tyrl71Pro 4 Thr215Asp

Ser2lβAsp Ser 70Asp 4 Tyrl71Ser 4 Thrl95Gly 4 Tyr210Asp 4

Ser220Glu Thrll3Gly 4 Glyl62Ser 4 Trp20δGlu 4 Ser220Asp +

Thr224Asp Ser 70Glu + Asnl40Ser 4 Ala203Gln 4 Ala219Glu 4

Gly223Pro Trpll2Pro + Asnl70Ser 4 Ser216Asp + Thr217Asp 4

Ser220Asp Thr 73Asn 4 Vall38Asp 4 Prol69Glu 4 Asnl70Ser 4

Thr217Gly Ser 70Asp + Aspl05Glu + Glyl62Pro + Trp206Val +

Thr217Ser Gly 67Asn 4 Asnl40Ser 4 Ala219Glu 4 Leu221Met 4

Ser222Glu Gly 72Ser 4 GlyllOPro 4 Trpll2Ala 4 Tyr210Asp 4

Thr217Glu GlylOδPrc 4 Trpll2Leu + Leul34Cys 4 Glyl35Asp 4

Alal64Asp Asn ββGln 4 Trp20δAla 4 Thr217Glu 4 Ser220Glu 4

Leu221Thr Thr 73Gly Leul34Ser 4 Thr217Glu 4 Ser220Glu 4

Leu221Asn Gly 67Ser 4 AlalβδGly + Pro214Gln 4 Thr217Glu +

Ser220Asp Valll5Cys 4 Glyl39Asn 4 Tyr210Cys 4 Thr217Asp 4

Ser220Asp Leul34Glu 4 Thrl37Glu 4 Thrl95Asn 4 Trp20δSer +

Leu221Ser Alal64Gln 4 Tyr210Val 4 Thr215Asp 4 Thr217Glu 4

Ser220Asp Gly β7Asp 4 Ser 70Asp 4 Thr217Gly 4 Tyr218Asp +

Leu221Ile Ser 70Asp 4 ThrlllAsn 4 Trp208Cys 4 Thr217Glu 4

Ser220Glu Leul34Glu 4 Glyl62Ser 4 AsnlββGln 4 Ser207Asp +

Gly223Asp Glyl62Pro 4 Thrl67Asn 4 Prol69Glu + Serl91Glu 4

Phel93Asp Ser 70Glu 4 Trpll2Tyr 4 Ser207Glu + Tyr216Cys 4

Leu221Glu Asn ββGlu 4 Leul04Glu 4 Serl07Asp 4 Trp20δSer 4

Thr215Pro Leul04Ala 4 Glyl35Asp 4 Thrl95Asp 4 Tyr213Ile 4

Thr217Gly Thrl67Gln Serl92Glu + Ser207Glu 4 Trp20δCys 4

Ser220Asp Glyl65Pro 4 Serl91Asp 4 Trp20δGly 4 Ser220Asp 4

Leu221Glu Glyl35Glu 4 Asnl63Asp + Tyr213Met 4 Leu221His +

Ser222Asp ThrlllAsp 4 Glyl36Glu 4 Glyl62Glu 4 Alal64Asn 4

Pro214Ser Thrll3Gln Tyr213Thr + Ser216Glu + Thr217Glu +

Leu221Glu Ser 70Glu 4 Glyl08Glu + Alal64Gly 4 Glylβ5Gln 4

Leu221Asp Ser 70Glu 4 Leul04Gln 4 Ser220Asp 4 Leu221His 4

Gly223Pro Glyl65Asρ 4 Prol69Ser 4 Ser207Glu 4 Trp208Gly +

Ser222Asp AlalβδPro 4 Prol69Asn 4 Thr215Asp 4 Thr217Asp Leu221Glu GlyllOPro Thrll3Ser 4 GlylβSGlu + Trp208Asp 4

Ser222Asp GlyllOGlu 4 Thrll3Asp 4 Glyl39Glu 4 Trp208Tyr 4

Tyr210Ala Asn βδGlα 4 AsnlOβGlu 4 Tyrl71Hιs 4 Tyr210Gln 4

Ser216Glu Alal64Glu AlalβδHis 4 Ala219Glu 4 Ser220Asp 4

Leu221Cys Asn βδAsp 4 Tyr210Gln 4 Ala219Glu Ser220Glu 4

Leu221Ser Thr 73Asp 4 Asnl63Ser 4 Ala219His 4 Ser222Glu 4

Gly223Glu Gly 67Asp 4 Asn 68Glu + AlalβδGln 4 Prol69Glu 4

Phel93Asn GlyllOAsp 4 Glyl39Gln 4 Asnl40Glu 4 Serl41Glu 4

Tyr216Asn Serl09Glu 4 Ser216Glu 4 Thr217Asp 4 Tyr21δAsn 4

Leu221Hιs Glyl35Pro 4 Glyl36Ser 4 Phel93Asp 4 Ser216Asp 4

Thr217Glu Glyl65Gln 4 Serl92Glu 4 Ala203Ser 4 Ser216Asp 4

Thr217Asp Gly 67Gln 4 Leul34His 4 Glyl36Asp 4 Ser207Asp 4

Trp20δGlu Vall03Ser 4 Ser207Asp 4 Trp20δGlu + Thr217Glu 4

Leu221Cys Gly 69Pro + Aspl05Glu 4 AsnlOβAsp 4 AsnlββAsp 4

Ile209Thr Trpll2Tyr 4 Asnl70Glu 4 Trp20δGln 4 Ser220Glu

Leu221Glu Thrl37Gly 4 Glyl39Gln + Glyl62Asp 4 Ser220Glu

Leu221Asp Thrll3Asp 4 Alal64Gly + Serl91Glu 4 Serl92Glu 4

Thrl95Gly Serl07Glu 4 Thrll3Glu 4 Alall4Asp 4 Tyr210Pro 4

Leu221Ala Asn ββAsp 4 Gly 67Asp 4 Leul34Pro 4 Tyr210Asn 4

Ser222Asp Asn βδGln + Leul04Ala 4 Prol69Asp 4 Asnl70Glu 4

Leu221Asp AsnlOβGlu 4 Serl07Asp 4 Glyl39Asp 4 Ala203Asn 4

Thr217Ser Thr 73Gly 4 Thrl37Glu 4 Tyr210Asp 4 Thr217Gln 4

Ser220Glu Trpll2Ile 4 Glyl36Glu Phel93Thr + Tyr210Glu 4

Ser220Glu Trpll2Ala 4 Asnl63Ser 4 Serl92Glu 4 Tyr210Glu

Ser220Glu Ser 70Glu + Thr 73Ser 4 Vall03Asp 4 Pro214Glu 4

Tyr216Gly AsnlOβGlu Serl09Glu 4 Thrl95Gln 4 Trp20δPro +

Pro214Glu Asn βδGin + Vall03Asp + Thrl37Gln 4 Ser207Asp +

Ser222Asp Thrl37Asp 4 Ser207Glu 4 Thr217Gly 4 Leu221Pro 4

Ser222Asp Gly 69Asp 4 Thr 73Glu + ThrlllPro + Glyl39Asn 4

Ser220Asp Asn ββGlu 4 Ser 70Asp + Pro214Gly 4 Ser220Glu 4

Leu221Ser Ser 70Glu 4 Leul04Cys 4 Tyr210Ser 4 Thr215Glu +

Ser220Glu Thrl67Pro 4 Ser216Glu 4 Ala219Glu 4 Leu221Met 4

Ser222Asp Vall36Asp 4 AsnlββGlu 4 Thrl67Asp + AlalβδThr

Leu221Ala Leul34Pro 4 Serl41Glu 4 Trp206Glu 4 Thr217Ser 4

Ser220Glu Glyl35Glu 4 Tyrl71Asn 4 Pro205Gln 4 Trp208Glu 4

Ser220Asp Trpll2Asn 4 Thrl67Glu 4 AlalβδGly + Trp20δAsp 4

Ser220Glu Asnl40Asp + Prol69Gln 4 Trp206Glu + Thr215Gln 4

Ser220Glu AsnlOβGln 4 Glyl36Asp 4 Trp206Glu + Thr217Ser 4

Ser220Glu AsnlOβAsp 4 ThrlllAsn 4 Thrl67Gly 4 Tyr210Asp 4

Ala219Asp Asn ββGln 4 Prol69Asp + Tyrl71Glu 4 Pro214Asn 4

Gly223Glu Trpll2Gly Asnl63Asp + Thrl67Glu 4 Serl91Glu 4

Gly223Ser Trpll2Asp 4 Thrll3Ser 4 Alall4Glu 4 Thr217Glu 4

Gly223Gln Asn ββAsp 4 Thr 73Gly Vall03Asp 4 ThrlllPro 4

Thrl95Asn Asn 68Asp 4 Trpll2Met + Trp208Asn 4 Pro214Asp 4

Ser220Asp Aspl05Glu Serl09Glu + Asnl63Glu 4 AsnlββSer

Tyr21δGly GlyllOGln Trpll2Asp + Leul34Pro + Pro214Asp 4

Thr217Asp Gly 67Pro 4 Thrll3Glu + Glyl35Asp Glyl62Asp 4

Thr215Gln Ser 70Glu 4 Thrl95Ser 4 Pro214Ser + Ser220Glu 4

Gly223Glu GlyllOPro Vall3δGlu 4 Trp20δGlu Tyr210Glu

Thr217Gly GlylOδAsn 4 Asnlβ3Glu + Ala219Asp 4 Leu221Val +

Thr224Asp Thr 73Asp GlylOδAsn + Serl09Glu + Pro214Glu +

Thr217Asn Thrl37Asp 4 Glyl39Glu + Asnl70Ser + Trp208Cys

Tyr210Asp Trpll2Phe Glyl39Glu + Tyr213Thr 4 Ala219Glu + Leu221Glu Leul04Pro 4 Prol69Glu 4 Tyr210Glu 4 Leu221Asp 4

Thr224Gly AsplOSGlu 4 Glyl65Glu 4 Thrl67Gln 4 AlalβδGlu 4

Tyr210Pro Thrl37Asp + Trp20δAsn 4 Tyr216Ser 4 Ser220Asp 4

Ser222Asp Gly 69Glu 4 Thrll3Gln 4 Thr217Ser 4 Ser220Glu 4

Ser222Glu Leul04Hιs 4 Thr::3Asp 4 Ser207Asp 4 Thr2:7Ser 4

Leu22:Glu Alai:4Thr + G:y:35Asp 4 Ser207Asp 4 Thr217Ser 4

Leu22:Asp Gly 69Ser 4 Leul04Asp 4 AsnlOβGlu 4 Serl91Glu 4

Thr217Gly Vall03Ala + Tyrl71Val 4 Ser207Asp 4 Ser216Asp 4

Tyr21δAsp Gly 69Ser 4 Leul34Ile 4 Glyl62Asp 4 Ser207Glu 4

Ser220Glu Gly 72Pro 4 Thrll3Glu + AsnlββAsp 4 Thrl67Asp 4

Thr217Gln AsnlββGlu 4 Thrl67Asp 4 Ser207Glu 4 Thr217Gly 4

Leu221Val ThrlllAsn 4 Trpll2Glu + Vall36Gly 4 Asnl63Asp 4

Tyrl71Glu Gly 67Pro 4 AsnlOβGlu + Leul34Pro 4 Thrl37Asp +

Prol69Glu Thrl95Glu 4 Ser207Asp 4 Ser220Asp 4 Leu221Val +

Thr224Gly Asn βδAsp + Gly 69Gln 4 Glylβ2Asp 4 Prol69Ser 4

Gly223Asp Thr 73Ser 4 Glyl65Asn 4 Ser207Asp 4 Tyr210Asp 4

Ser216Glu Vall03Glu Alal64Asp 4 Asnl70Glu 4 Trp208Ala +

Tyr210Ile Alall4Thr 4 Alal64Asp 4 Asnl70Glu 4 Tyr210Ala 4

Thr215Asp Asn 68Glu 4 Glyl39Pro 4 Alal64Pro 4 Serl91Asp 4

Pro214Glu Asn 68Glu 4 Alal64Glu 4 Glylβ5Ser 4 Tyr210Gly 4

Pro214Asp Gly 67Ser 4 Thrl67Gly + Ser207Glu 4 Ser216Asp 4

Ser220Asp Aspl05Glu 4 GlyllOAsn 4 Ser207Asp 4 Ala219Gly 4

Ser220Glu Vall03Gly + Asnl40Asp 4 Ser207Asp 4 Tyr213Asn +

Ser220Asp Serl09Glu 4 Glyl65Asn 4 Ser207Glu 4 Tyr210Cys 4

Ser220Asp GlyllOAsn 4 Asnl40Glu 4 Ser207Glu 4 Thr217Ser 4

Ser220Glu GlyllOAsp + Glyl36Pro 4 Gly206Gln 4 Ser207Glu 4

Ser220Glu Thrll3Gln 4 Leul3411e -*^■ Serl92Glu 4 Thrl95Asp ^■>^■

Ser220Asp Glyl36Glu 4 Trp206Glu 4 Thr217Gly 4 Leu221Asn 4

Gly223Asp Serl07Asp + GlyllOAsp 4 Leul34Ser 4 Asnl40Glu 4

Tyr210Thr Thrl67Glu 4 Trp206Asp 4 Thr217Asn 4 Ala219Asp 4

Gly223Asn Gly 69Glu + Alall4Asp 4 Thrl95Ser 4 Trp203Ser 4

Pro214Glu Vall03Asp 4 Thrll3Glu 4 Phel93Tyr 4 Trp20δAsp +

Ala219Pro AsnlOβAsp 4 Glyl36Ser 4 Vall38Met 4 Serl91Asp +

Ser222Asp Glyl35Asp 4 Glyl39Glu -<- Tyr210Ala 4 Thr217Ser 4

Ser222Asp Thrll3Asp 4 Asnl40Ser 4 Ala203Gln 4 Ser207Glu 4

Tyr210Glu GlylOδGlu 4 Thrll3Gln 4 Ser207Glu 4 Tyr210Asp 4

Pro214Asn Glyl35Glu 4 Asnl63Asp 4 Tyr210Met 4 Tyr21δPro 4

Ser220Asp Asnl63Asp Alal64Thr 4 Serl91Glu 4 Thr217Asp

Thr224Gln Asn βδGlu 4 Leul04Asp 4 Valll5Thr 4 Alal64Asp 4

Pro214Gln Thr 73Gly 4 Serl07Glu 4 GlylOδPro 4 Leu221Glu 4

Thr224Asp Gly 67Ser 4 Glyl62Asp + Thrlβ7Asp 4 Ser207Glu 4

Thr217Ser Gly 67Gln 4 Serl09Glu 4 Vall38Ser 4 Pro214Glu 4

Ala219Glu Serl07Glu 4 Glyl35Glu + Trp208His + Thr217Ser 4

Thr224Glu Asn ββGlu 4 Aspl05Glu Vall38His 4 Serl92Glu +

Tyr210Ile Asn ββGlu 4 Aspl05Glu 4 Ala219Gln + Ser220Glu +

Leu221Asn Gly β9Ser 4 Ser 70Glu 4 GlyllOGlu + Thr217Asp 4

Leu221Ala Tyrl71Glu Serl92Asp 4 Ser207Glu + Trp208Pro 4

Tyr210Met Gly β7Gln 4 Aspl05Glu 4 Alal64Asp 4 Prol69Asn 4

Gly223Asp Thr 73Asp + Serl41Asp + Trp20δLeu + Ser216Glu +

Thr217Pro Asn ββAsp + Trp208Gln 4 Ile209Thr 4 Tyr210Val +

Ser216Glu Asn ββSer 4 Vall03Glu 4 AsnlOβAsp + Serl91Glu +

Tyr210Ala Asn 68Gln Leul34Glu 4 Serl92Glu + Leu221Gly +

Ser222Glu Ser 70Asp 4 Aspl05Glu 4 GlylOδSer 4 Glyl39Glu + Thr217Gly Gly 67Ser 4 AsnlOβAsp 4 Glyl65Gln 4 Ala219Asp 4

Ser222Glu AsnlOβGln - Leul34Met 4 Asnl40Asp 4 Ala219Asp 4

Ser222Asp Gly 69Giu 4 Vall03Gln 4 Leul34Asp + Thrl67Gln 4

Trp20δGly Thrl37Glu 4 Glyl65Glu 4 Tyr210Ser 4 Thr217Ser 4

Leu221Pro Serl07Asp 4 Glyl62Asp 4 Thrl67Asp 4 Tyrl71Cys +

Trp20δlle AsnlOβGlu 4 Trpll2Asp 4 Leul34Gly 4 Serl41Glu 4

Phel93Ser Alail4Thr 4 Asnl40Asp 4 Phel93Thr 4 Thr217Giu 4

Ser220Glu Thrll3Ser 4 ValllSAsp 4 Leul34Val 4 Thr217Asp 4

Ser220Glu GlyllOAsp 4 Asnl70Gln 4 Thr217Glu 4 Tyr21δAla 4

Ser220Asp Thrl67Asp 4 Phel93Thr 4 Thr217Asp 4 Ala219Gln 4

Ser220Asp GlylOδGlu 4 Pro214Asn 4 Thr217Glu + Ser220Asp 4

Leu221Val AsnlOβGln 4 Glyl35Glu 4 Vall36His 4 Thr217Glu 4

Ser220Glu GlyllOGlu 4 Tyr210Met 4 Thr217Glu 4 Ser220Asp 4

Leu221His Gly 67Glu + Serl07Glu 4 GlyllOSer 4 Thrll3Glu 4

Thr215Ser Gly 67Glu 4 Serl07Glu ThrlllPro 4 AlalβδHis +

Ser220Glu ThrlllAsp 4 Glyl36Asp 4 Trp208Glu 4 Tyr210Met 4

Thr217Gln Aspl05Glu 4 Glyl62Ser 4 AsnlββAsp 4 Serl91Asp 4

Trp208Phe Aspl05Glu 4 AsnlββGlu 4 Serl91Glu 4 Trp20δAla 4

Leu221His Gly 67Glu 4 Vall03Glu 4 Alal64Asn 4 Ser207Asp 4

Tyr218Ala Gly 72Asn 4 Leul04Asp 4 AsnlOβGln 4 Thrll3Asp 4

Thr217Asp Ser 70Asp 4 Glyl35Glu + Alal68Glu 4 Tyr210Ile 4

Ala219Gly AsnlOβGln 4 Serl09Glu 4 Thrll3Asp Thrl37Glu 4

Trp208Asn Alall4Ser 4 Vall38Thr 4 Prol69Asn 4 Phel93Asp

Trp206Glu Asn 68Asp + Vall38Asp + Alal64Gly 4 AlalβδGlu 4

Thr217Gly Gly β9Asn 4 Vall3δAsp 4 AlalβδGlu 4 Ser207Asp 4

Thr217Pro GlyllOSer 4 ThrlllGln 4 Alal64Glu 4 Thr215Ser 4

Ser222Asp Leul04Ser 4 GlylOδGlu 4 Valll5Glu 4 Vall38His 4

Serl41Glu Ser 70Glu 4 Alall4Ser Thrl37Ser 4 Ser207Asp 4

Ser21βAsp Gly 69Asp 4 GlyllOAsp 4 Thrl37Glu 4 Prol69Ser 4

Tyrl71His Ser 70Asp + Thrl37Glu 4 Alal64His 4 Trp20δlle +

Ser220Glu Ser 70Glu 4 Thrl37Glu 4 Thr217Asn 4 Ser220Glu 4

Gly223Asn GlyllOGln 4 Trpll2Cys 4 Valll5Glu 4 Asnl40Asp +

Ser216Glu Asn βδAsp 4 Serl09Glu AsnlββGln 4 Trp20δPro +

Leu221Asp Thrll3Pro 4 Thrl67Glu 4 Tyrl71Glu 4 Thr217Gln +

Ser220Glu Trpll2Ile 4 Leul34His + Glyl39Asp 4 Glyl62Glu +

Trp206Val GlyllOAsp 4 Glyl62Gln + Trp206Ile 4 Tyr210Glu 4

Gly223Asp Gly β7Asp 4 ThrlllAsp Trp206Val Tyr210Ser 4

Ser216Glu Gly 69Asn 4 Thr 73Glu 4 AsnlOβAsp 4 Serl41Glu +

Glyl62Asn Gly 69Glu 4 Glyl35Asn 4 Prol69Asn 4 Trp20δAla +

Thr217Glu Ala203Gly + Trp206Ala 4 Pro214Glu 4 Ala219Gln 4

Leu221Asp Thr 73Gly GlyllOGlu Pro214Asp 4 Ser220Glu +

Leu221Asn Vall03Ala + Thrl67Gly 4 Serl92Glu 4 Pro214Glu 4

Ser220Asp Aspl05Glu + Tyrl71Gln 4 Pro214Asp + Thr217Ser +

Ser220Asp Tyrl71Val 4 Serl91Asp 4 Trp208Pro Pro214Glu 4

Ser220Glu Gly β7Asp + Glyl62Ser 4 Gly206Ser 4 Ser207Glu +

Thr217Glu Gly 67Glu 4 Trpll2Phe + Ser207Asp 4 Thr217Asp

Ala219Gln Asn βδAsp Asnl63Glu 4 Phel93Met Gly206Ser

Leu221Glu Asn ββAsp + Leul04Ala + Serl09Glu 4 AlalβδThr 4

Ser216Glu Vall36Met + Glyl39Asp 4 Glyl62Ser 4 AlalβδGlu 4

Ser220Asp

TABLE36

Multi-loop Sextuple Mutation Variants ββGln Gly β7Ser 4 Gly 69Ser 4 Leul04Gly + Tyr210Pro + Thr217Glu Ser 7OAsp 4 GlyllOPro 4 Glyl36Gln 4 Phel93Ser 4

Trp20δCys 4 ^~Leu221His Thrll3Pro Giyl39Asn 4 Serl41Glu 4 Tyrl71Pro 4

Trp208Thr + Leu221Gly Gly 69Gln 4 Leul04Thr 4 Serl07Asp 4 Leul34Thr 4

Thr215Ser 4 Leu221Gly Leul04Met 4 Leul34Ala + Thrl37Asp 4 Trp20δGly 4

Tyr210Val + Pro214Gly Gly β9Ser 4 Thr ^"'SAsp 4 GlyllOSer 4 Tyr210Leu 4

Thr215Gly + Thr217Ser Asn ββSer + Gly 72Gln 4 Thr 73Pro 4 GlyllOAsp 4

Trp206Val + Leu221His ThrlllAsn 4 Alal64His 4 Thrlβ7Glu 4 Phel93Met

Tyr210Asn 4 Thr217Gln Asn ββGln + Thr 73Gly 4 Thrll3Asp + AsnlββSer 4

Thrl67Pro 4 Tyr210Pro Leul04Met 4 Pro205Gln 4 Ser207Asp 4 Trp20δAla 4

Thr212Gly 4 Thr217Pro Leul34Val 4 Giyl65Gln 4 Trp206Thr 4 Pro214Gly 4

Leu221Ile 4 Thr224Asp Asn ββSer 4 Serl09Asp 4 Alall4Asn 4 Valll5Asn 4

Trp20δGln + Tyr210Ser AsnlOβGln 4 Alal64Pro 4 Glyl65Ser 4 Tyrl71Ala 4

Ser220Glu 4 Thr224Ser GlyllOSer 4 Glyl36Pro 4 Ile209His 4 Thr217Gln 4

Leu221Ile 4 Thr224Asp Asn ββGln 4 Valll5Pro 4 Trp20δAsp 4 Thr217Ser 4

Leu221Ile 4 Thr224Pro Gly 67Glu 4 Thr 73Asn 4 Asnl70Ser + Tyr210Ser 4

Thr212Gln 4 Gly223Pro Vall03Gly 4 Alal64Thr 4 Tyr210His + Ala219His 4

Ser220Glu 4 Leu221Gly Thr 73Pro 4 Asnl40Gln 4 Thrlβ7Asp 4 Trp20δMet 4

Thr217Gln 4 Leu221Asn Serl09Asp + Alal64His 4 Tyr210Pro + Thr212Ser 4

Gly223Pro + Thr224Asn GlyllOGln 4 Tyr210Pro 4 Tyr213Thr + Tyr21δMet +

Ser220Asp Leu221His Vall3δPro 4 AlalβδGly 4 Prol69Gln 4 Tyr210Ser 4

Thr217Gln 4 Ser220Asp Alall4Thr 4 Thrl37Asn 4 Glyl39Ser 4 Trp20δPro 4

Pro214Gln 4 Ser220Glu Vall03Met 4 Thrl37Ser 4 Ala203Ser 4 Tyr210Thr 4

Leu221Met 4 Gly223Ser Leul04Glu 4 ThrlllSer 4 Valll5His 4 Glyl65Asn 4

Tyr210Met 4 Ala219His GlyllOPro + Leul34Pro 4 AsnlββAsp 4 Asnl70Ser 4

Trp20δAsn 4 Thr217Asn Thrl67Glu + Prol69Gly 4 Asnl70Gln + Trp20δGly 4

Thr215Ser 4 Leu221Ser ThrlllSer 4 Thrl37Ser 4 Thrl95Gln 4 Ser207Glu 4

Tyr210Val 4 Thr217Pro AsnlOβAsp 4 Glyl36Pro 4 Glyl62Ser 4 Trp20δlle 4 Tyr210Gly 4 Pro214Gln Ser 70Asp 4 Glyl65Gln 4 Phel93Asn 4 Thrl95Ser 4

Trp20δPro Thr212Ser Gly 72Ser 4 Alall4Gln AsnlββGln 4 Phel93Glu 4

Tyr210Gly Thr224Pro Asn ββSer 4 Glyl36Ser 4 Glyl62Pro 4 Asnl63Ser +

Alal64Asp 4 AlalβδGln Gly 67Pro AsnlOβGln 4 Serl91Asp 4 Trp208His 4

^"τyr21δHιs 4 Leu221Asn Trpll2Leu 4 AlalβδPro 4 Trp20δHis + Tyr210Ser 4

Thr217Gly Ser220Asp Asn ββSer 4 ThrlllGly 4 Valll5Gly 4 AlalβδSer 4

Asnl70Gln 4 Pro214Gly GlyllOSer 4 Trpll2Val 4 Glyl62Asn 4 Thrlβ7Pro

Prol69Gln 4 Ser2lβAsp Gly 69Asn 4 AsnlOβSer 4 Glyl39Glu 4 Asnl70Ser 4

Tyr210Cys 4 Leu221Gly Gly 69Asn 4 Serl41Asp 4 Phel93Asn 4 Tyr210Ile 4

Ala219Asn 4 Leu221Ser Serl09Asp 4 Prol69Ser 4 Trp20δHis 4 Pro214Asn 4

Thr215Asn 4 Thr217Gly Asn βδAsp 4 Glyl62Gln 4 Alal64His 4 Trp20δPro 4

Tyr210Leu 4 Leu221Val Vall03Thr 4 Glyl39Pro + Alal64Thr 4 Thr217Ser 4

Leu221Pro 4 Thr224Pro Gly 69Glu 4 Gly 72Gln 4 Thrl37Pro Trp208Ile 4

Tyr21δPro 4 Gly223Gln Gly 67Pro 4 Leul04Cys 4 Vall38Met 4 Glyl39Ser 4

Ala203Gln 4 Ala219Asn Trpll2Ser 4 Phel93Ala + Thr217Gly 4 Tyr21δGlu 4

Leu221Met 4 Thr224Asn Asnl63Gln 4 Trp20δLeu 4 Tyr210Ser 4 Thr217Glu +

Ala219Gly 4 Gly223Pro Vall03His 4 GlyllOGln Glyl39Ser 4 Serl92Asp 4

Phel93Asn 4 Thr217Gln GlylOδGln 4 Prol69Asn 4 Thrl95Ser 4 Trp20δAla 4

Thr215Pro 4 Thr217Glu Serl09Asp 4 ThrlllAsn 4 Trpll2Gln 4 Thrl67Gln 4

Thrl95Gly 4 Tyr210Asn Gly 72Gln 4 Trpll2Leu 4 Glyl36Asp 4 Tyrl71His 4

Tyr210Thr 4 Leu221Ser Gly 69Glu 4 Alal64Pro Trp208Val + Tyr210Thr 4

Tyr218Gln 4 Thr224Ser Alall4Pro + Leul34Glu + Asnl40Ser + Alal64Gln 4

Phel93Ile 4 Trp206Asn Asn ββSer + Vall38Met + Glyl39Asn + Asnl63Gln 4

Glylβ5Gln 4 Tyr210Thr AsnlOβGln + Serl91Asp 4 Phel93Leu 4 Trp20δVal 4

Pro214Gln 4 Thr217Ser Alall4Thr 4 AsnlββSer 4 Tyrl71Gly 4 Serl91Glu +

Ala203Pro 4 Tyr210Ala Asn βδGln 4 Tyrl71Asp Phel93Tyr 4 Gly206Ser 4

Tyr210Val 4 Leu221Thr Leul04Aia 4 Alall4Ser 4 Asnl63Asp 4 Thrl95Gly 4

Gly206Gln 4 Leu221Ala GlyllOAsn 4 Valll5Asn -^ Trp20δCys Tyr210Hιs 4

Ala219Thr + Gly223Asp Asn ββAsp Gly 69Asn 4 Valll5Ala 4 Alal64Ser 4

Trp206Gln 4 Thr217Gln Valll5Met Thrl37Ser 4 Glyl62Asn 4 AsnlββGlu 4

Tyrl71Hιs 4 Pro214Ser AsniOβGln 4 GlyllOGln 4 Trp208Hιs 4 Thr217Pro 4

Ala219Asp 4 Ser220Asp Gly 67Asn 4 Gly 72Asn 4 Alall4Gly 4 Asnl40Gln 4

Ser216Asp -r Thr217Asp Gly 69Gln 4 Thr 73Ser 4 Prol69Gln 4 Trp208Ile 4

Ser220Glu 4 Leu221Asp Asnl70Glu 4 Tyrl71Asp 4 Phel93Ser Trp20δHis 4

Leu221Thr 4 Thr224Gly Gly 67Ser 4 Gly 69Glu + Ser 70Glu 4 Vall03Gly 4

Tyr210Gln 4 Ala219Asn Trpll2Gly 4 AsnlββSer 4 Trp20δMet 4 Tyr210Glu 4

Ser220Glu 4 Thr224Asn Leul34Pro 4 Thrl95Gly Trp20δGlu 4 Tyr210Asp 4

Thr217Ser 4 Ser220Glu Leul04Gln 4 Trpll2Gln 4 Tyrl71Pro 4 Trp208Glu 4

Thr217Gly 4 Leu221Glu Aspl05Glu 4 GlylOδAsp 4 Glyl36Gln 4 Thrl67Gln 4

Tyrl71Gln + Leu221Val Serl07Glu 4 Serl09Glu 4 Vall3δMet 4 AlalβδHis 4

Prol69Gln 4 Phel93Ser Gly 67Glu 4 Ser 70Glu 4 Trpll2Hιs 4 Tyrl71Ile 4

Trp208His 4 Tyr210Ala Vall03Pro 4 Trp208Glu 4 Tyr210Ser 4 Ala219Glu 4

Ser220Glu 4 Ser222Asp Serl91Asp 4 Ser207Glu 4 Tyr210Ala 4 Thr215Gly 4

Thr217Gln 4 Gly223Ser AsnlOβGlu 4 Serl09Asp 4 AlalβδThr 4 Tyrl71Met 4

Phel93Ile 4 Trp206Leu Asn βδSer 4 Thrl37Gln 4 Serl91Asp 4 Ser207Asp 4

Trp206Asp 4 Leu221Thr Vall03Gln 4 Vall3δHιs 4 AsnlββAsp 4 Serl92Glu 4

Trp20δHιs + Leu221Gly Vall03Pro 4 Glyl39Pro 4 Asnl63Asp Glyl65Gln 4

AlalβδGlu + Tyr210Leu Asn βδSer 4 Thrll3Gln + Leul34Ile 4 Serl91Glu 4

Leu221Hιs 4 Gly223Glu GlyllOGln 4 Ser207Glu 4 Trp20δGln 4 Thr217Pro 4

Tyr216Gly 4 Ser220Asp Glyl62Ser 4 Phel93Ala 4 Trp20δGlu 4 Ile209Ser 4

Leu221Cys + Gly223Glu Ser 70Asp 4 Aspl05Glu 4 GlylOδGlu 4 Asnl40Gln +

Trp20δLeu + Thr217Gly GlyllOAsn 4 Thrl37Glu 4 Prol69Asp 4 Asnl70Ser 4

Thrl95Glu + Tyr210Hιs Asn βδGln 4 Glyl62Glu 4 Alal64Thr 4 Glyl65Asp + Tyrl71Ala - Serl92Asp Vall36Met + Glyl39Asn 4 Asnl70Gln + Tyr210Glu 4

Thr21^"7Asp - Ser220Glu Glyl39Ser 4 Asnl63Asp + Alal64Ser + Serl91Asp 4

Tyr210Gly Leu221Ala Ser 70Asp 4 Thr 73Asp + Thrl67Ser + Trp206Ser 4

Thr217Asp 4 Ala219Asp Asn ββGlu 4 Leul04Glu 4 Aspl05Glu + Serl07Asp 4

Leu221Ser 4 Gly223Pro Asn ββGlu 4 Thr 73Asp + GlyllOPro + AlalβδGly 4

Tyr21δVal 4 Leu221Ile Gly 67Asn 4 Aspl05Glu 4 AsnlOβGlu + ThrlllGlu 4

Glyl65Asn 4 Thr217Gly Asn ββAsp 4 Tyr210Ser 4 Pro214Glu 4 Thr215Asp 4

Thr217Gln 4 Tyr218Asp Thr 73Asn + Serl92Glu + Ala203Thr 4 Ser207Asp 4

Trp20δGlu 4 Leu221Glu Vall38Gly + Prol69Glu + Serl92Glu + Thrl95Glu +

Trp208Pro 4 Thr212Gln Thr 73Asp 4 GlylOδAsn 4 Leul34Ser + Tyr210Ala +

Thr217Gln 4 Tyr216Glu Thr 73Asn 4 Alall4Thr + Vall38Ser 4 Glyl39Asp 4

Serl41Glu 4 Asnl70Glu GlylOδGln 4 Glyl35Pro 4 Glyl62Asn 4 Ser220Glu 4

Leu221Ala 4 Gly223Glu Vall03Gln 4 Trpll2Cys 4 Alal64Glu 4 Trp20δGln 4

Thr217Gly 4 Gly223Asp ThrlllGln 4 Leul34His 4 Serl91Asp 4 Phel93Asp 4

Thrl95Gln Trp2OδAsp Vall03Ser 4 Leul04Ile 4 GlyllOGlu + Glyl36Glu 4

Alal64Asn 4 Tyr210Gln Leul34Ser 4 Thrlβ7Gly 4 Serl91Glu 4 Ser220Asp 4

Leu221Pro 4 Ser222Asp Gly 69Gln 4 Trp2OδAsp + Pro214Glu 4 Ser220Asp 4

Ser222Glu 4 Gly223Asn Serl07Asp + Serl09Glu 4 Trpll2Pro 4 Glyl35Asp 4

AlalβδThr Thr212Gly Asnl40Ser + Thrlβ7Asp + Trp208Glu + Thr217Asn +

Ser220Asp Leu221Asp Glyl35Asp + Glyl62Glu Tyrl71Asp Tyr210Ala 4

Thr217Ser + Ser222Asp Valll5Asp + Tyr210Gln 4 Ser220Glu Leu221Asp 4

Ser222Asp 4 Gly223Ser Alall4His 4 AsnlββAsp 4 Tyrl71Cys 4 Tyr210Glu 4

Ser220Asp 4 Leu221Asp Valll5Cys 4 Glyl39Asn 4 Alal64Gly 4 Pro205Asn 4

Thr217Asp Ser220Asp Thrl67Gln 4 Tyr210Ile 4 Thr215Gly + Thr217Asp 4

Tyr218Asn 4 Ser220Asp Thrl37Ser 4 Trp208Leu + Tyr210Leu + Thr217Glu +

Ser220Asp 4 Leu221Gly ThrlllAsn + Glyl35Ser Trp2OδAsp + Tyr210Glu 4

Thr217Glu 4 Leu221Ala Thrll3Asn 4 Alall4Gly + Thrl37Asp + Thrl67Asp +

Trp206Gln 4 Gly223Asn Ser 70Asp 4 Thr 73G1U 4 Asnl63Asp + Thrl67Pro 4

Pro214Glu 4 Thr217Gly Glyl35Glu 4 Tyrl71Asn 4 Pro205Gln + Ser207Glu 4

Trp20δGlu 4 Ser220Asp Gly 69Asp + Aspl05Glu 4 GlylOδAsp + Ser207Asp 4

Trp20δThr 4 Gly223Asn Thrll3Gly 4 Alall4Pro 4 Tyr210Asp 4 Pro214Asp 4

Thr217Gln 4 Ser220Asp Asn βδGlu 4 Ser 70Asp 4 Aspl05Glu 4 Alall4Thr

Glyl35Ser 4 Ser220Asp ThrlllSer + Thrl37Glu 4 Ser207Glu 4 Trp20δThr 4

Ser220Glu 4 Leu221Asp Gly 69Asp 4 Leul04Asp + Aspl05Glu 4 Asnl40Glu

AsnlββSer 4 Leu221His Thrl37Asp 4 Glyl39Glu 4 Asnl70Glu 4 Trp20δCys 4

Tyr210Asp 4 Leu221Thr Gly 67Pro 4 Trpll2Gln 4 Serl92Asp 4 Tyr210Ile 4

Ser220Asp 4 Ser222Asp Vall03Gln 4 GlylOδAsp 4 Glyl35Glu 4 Thrl37Glu 4

AlalβδGln 4 Tyr210Asn Valll5Asp + Leul34Glu 4 AsnlββGln + Asnl7OAsp +

Tyr210Val 4 Thr212Pro Asn ββAsp 4 Asn βδSer 4 Thrl67Gln 4 Ile209Leu 4

Ser2lβAsp 4 Thr217Glu Vall03Glu 4 GlylOδAsp GlyllOGlu 4 Glyl65Pro 4

Thrl95Glu 4 Thr217Gln Asn ββAsp 4 Ser 70Glu 4 Thr 73Asp 4 Serl09Asp 4

Glyl36Pro 4 Tyr210Ile Gly 69Asp 4 Ser 70Glu + Aspl05Glu 4 Asnlβ3Glu 4

Tyr210Met 4 Thr217Gln Asn βδGln 4 Leul34Glu 4 Ser207Asp 4 Trp20δPro +

Tyr210Asp 4 Leu221Asp Asn ββSer 4 Valll5Gly + Glyl62Asp + Serl91Glu +

Trp206Ile 4 Thr217Ser Ser 70Glu 4 Tyr213Thr 4 Thr217Asn Tyr21δGly +

Ser222Glu 4 Gly223Asp Trpll2Met 4 Glyl65Asp Prol69Ser 4 Ser207Glu

Trp20δGly 4 Ser222Asp Gly 69Ser 4 Serl09Asp + GlyllOGlu 4 Alall4Gly 4

Glyl36Asp 4 Phel93Asp Alalβ4Gln 4 Glyl65Pro 4 Serl92Asp + Phel93Asp +

Ser207Glu 4 Thr217Asp Asn 68Glu 4 Thr 73Asn 4 Glyl62Gln + Trp208Gln

Ala219Glu 4 Ser220Asp Ser 70Glu 4 Leul34Ser 4 Glyl65Asp + AsnlββAsp +

AlalβδGly 4 Ile209Gly Leul04Asp 4 Aspl05Glu 4 Trp206Gly + Tyr210Glu 4

Tyr216Ala 4 Gly223Pro Asn βδAsp 4 Gly 69Glu 4 ThrlllAsn 4 Trpll2His 4

Glyl36Asn 4 Prol69Glu GlylOδSer + Asnl4OAsp 4 Serl41Glu 4 Alal64Gln 4 Ser207Glu 4 Tyr218Ser Leul04Cys 4 Glyl62Pro + Ser207Glu + Ser216Glu +

Thr217Asp + Ala219Thr Alal64Asn + AsnlδβAsp + Gly206Gln + Tyr210Thr +

Ser2iβAsp 4 Thr217Asp GlylϋoAsp 4 Serl09Asp AsnlββSer + Thr2l7Gln 4

Ser220Asp 4 Leu221Thr Serl09Asp 4 GlyllOAsp + Va::36Ala 4 Tyrl71His 4

Ala204His 4 Ser222Glu Asn 68Gln 4 Thrl37Gly 4 Prol69Asp 4 Ser207Glu 4

Trp208Glu 4 Tyr21δGln Trpll2Asp 4 Thrll3Glu + Vall36Gly + Serl91Glu

Trp20δPro 4 Leu221Ser Leul04Gly 4 Aspl05Glu 4 AsnlOβGlu + Gly20βSer +

Trp20δHis 4 Tyr218Glu GlylOδAsp 4 Leul34Asn 4 Glyl36Gln 4 Trp20δPhe 4

Ser220Asp 4 Leu221Asp GiylOδGln 4 Trpil2Tyr 4 Asnl70Glu 4 Trp20δGln 4

Ser220Glu 4 Leu221Glu Gly 72Asn 4 AsnlββAsp 4 Phel93Ser 4 Trp20δTyr 4

Ser220Glu 4 Leu221Glu Asn ββGlu 4 Gly 67Glu 4 ThrlllAsn 4 Thr215Gln 4

Thr217Asn 4 Ser220Glu AsnlOβAsp 4 Serl07Asp 4 Glyl65Asp + Phel93Val 4

Trp208Val 4 Leu221Thr Leul04Val 4 Glyl65Gln 4 Serl92Asp 4 Phel93Glu 4

Tyr210Glu 4 Leu221Cys Glyl36Gln 4 Prol69Ser 4 Serl92Asp 4 Phel93Asp 4

Trp208Leu 4 3er220Glu Serl07Asp 4 Valll5Thr 4 Thrl37Gln + Glyl62Asp +

Asnlβ3Asp 4 Ala203His Vall03Met + Serl07Glu 4 Trp206Val + Thr215Glu +

Ser21βGlu 4 Gly223Ser Vall03Ser 4 Asnl4OAsp 4 Serl41Glu 4 Tyr210Glu

Ser220Asp 4 Gly223Asn Asnlβ3Asp + Asnl70Glu 4 Thrl95Asn 4 Ser207Asp 4

Tyr210Met 4 Tyr218Pro Asn ββAsp 4 Glyl39Gln 4 Tyr210Glu + Thr217Ser 4

Ser220Asp 4 Thr224Asn Gly 69Asp 4 AsnlOβAsp 4 GlyllOAsp + Glyl65Asn 4

Prol69Ser 4 Thr217Asp Phel93Ile 4 Thr215Ser 4 Thr217Glu 4 Ser220Asp 4

Leu221Pro 4 Gly223Glu Vall03Gly 4 Aspl05Glu 4 Thrl95Asn 4 Ser207Asp 4

Ser222Asp 4 Gly223Asn Gly 69Asp Asnl40Gln 4 Alal64His + AlalβδSer 4

Ser207Asp 4 Ξer222Asp Asn 68Asp 4 ThrlllAsn 4 Alall4Gly 4 Glyl35Gln 4

Ser207Glu 4 Ser222Glu AsnlOβAsp 4 Glyl39Asn 4 Glyl62Glu 4 Prol69Ser 4

Serl91Asp 4 Thr224Asp Thrll3Asp + Serl92Asp + Ser207Glu + Tyr213Thr

Thr215Pro 4 Ser222Asp Gly 69Pro Asnl63Asp + Thrl95Glu 4 Trp206Ser 4

Tyr210Glu 4 eu221Cys Gly 69Pro Ser 70Asp 4 GlyllOSer 4 Glyl35Glu 4

Asnl70Glu 4 Leu221Ile Gly 69Glu - Alal64His + Thrl95Gln 4 Ser220Glu 4

Leu221Glu 4 Thr224Glu Gly 69Gln Glyl36Asp 4 Val13δAsp 4 Prol69Gly 4

Asnl70Gin 4 Ser220Glu Glyl36Glu + Vall3δAsp 4 Thrl95Asn 4 Ser220Glu 4

Leu221Gly 4 Gly223^"pro Asn βδGln 4 Serl07Glu 4 Serl09Glu 4 Glyl39Gln 4

Alal64Gln 4 Ser207Asp Serl07Glu + Serl09Glu 4 Prol69Gln 4 Tyr210Met 4

Thr217Pro 4 Ser222Asp Gly 67Asp 4 Ser 70Glu 4 Vall03Ala 4 ThrlllGlu 4

Tyr210Asn 4 Pro214Ser Asn ββGlu 4 Gly 67Asp 4 Leul34His + Serl91Asp 4

Ser207Glu 4 Trp206Thr Asn ββGlu 4 Vall03Asp 4 GlylOδAsn Asnl70Ser 4

Thr217Asn 4 Leu221Met Thr 73Glu + Trpll2Pro 4 AsnlββSer 4 Phel93His 4

Tyr21δlle 4 Ser220Glu Valll5His 4 Ser207Glu 4 Trp20δTyr 4 Tyr210Leu 4

Pro214Glu 4 Leu221Glu Asn ββGln 4 Serl07Glu 4 Serl09Glu 4 Ser207Glu 4

Gly223Asp 4 Thr224Gly Asn ββGlu 4 Asn βδAsp 4 Thr 73Gln 4 Thrl95Ser 4

Ser207Asp 4 Tyr216Pro Ser 70Asp 4 Leul04Ile 4 Asnl63Asp 4 Thr217Gln 4

Leu221Val 4 Thr224Glu Gly 67Asp 4 Vall03Glu 4 AsnlOβAsp 4 AlalβδAsn 4

Thrl95Glu 4 Trp20δLeu Thrl37Gly 4 Glyl62Glu + Phel93Asp 4 Trp206Asp 4

Thr217Asn 4 Tyr21δVal Glyl35Pro 4 Glyl39Glu 4 Ser207Glu 4 Trp206Ala 4

Tyr210Cys 4 Gly223Glu AsnlOβSer 4 Thrl37Asp 4 Glyl39Glu + Serl41Asp +

Ala204Gln 4 Trp206Glu Trpll2Ala 4 Asnl63Glu + Serl92Glu 4 Trp206Val 4

Tyr210Glu 4 Ser220Glu GlyllOAsp 4 Trpll2Asp 4 Tyr210Thr + Ser220Glu 4

Leu221Met 4 Ser222Glu Gly 67Asp 4 Gly 69Asp 4 Thr 73Pro + Serl09Asp 4

Trp206Gln 4 Ser222Asp Asn ββSer 4 Vall03Glu 4 Aspl05Glu 4 Trpll2His 4

Serl91Glu 4 Tyr210Ala Gly 67Glu 4 Gly 69Asn 4 Glyl62Glu + Ser207Glu 4

Thr217Asn 4 Gly223Glu Ser 7OAsp 4 Glylβ5Asp 4 AlalβδAsp 4 Trp20δPro 4

Thr217Gly 4 Leu221Ile Gly 67Glu 4 Vall03Asn 4 Glyl3βGln 4 Trp20δPro 4

Ser216Glu 4 Leu221Met Gly 67Asn + Glyl39Glu 4 Serl41Glu 4 Tyr213Gly + Ser220A_sp -*- Leu221Asn Gly β7Giu 4 Leul04Ser 4 Tyr210Ser + Ser220Asp +

Ser222Giu - Gly223Gln Gly 67Asp + Asn βδGlu + Glyl36Asp + AlalβδGln 4

Prol69Glu + Phel93Asn Asn ββGlu + Gly 69Asp + Asnl40Gln 4 Serl92Asp +

Phel93Asp 4 Tyr210Leu Leul04Asp + AsnlOβGlu 4 Asnl70Glu 4 Tyr210Ile 4

Thr215Ser 4 Thr224Pro Leul04Asp -- Serl07Glu 4 Glyl35Asp 4 Glyl39Asn 4

Alal64^~Glu 4 Thr217Gly Trpll2Glu 4 Giyl39Glu 4 Alal64Glu 4 Thrl95Glu 4

Thr212Ser 4 Tyr213Ser Asn 68Asp + ThrlllGly 4 Thrll3Ser 4 Val13δAsp 4

Asnl7OAsp 4 Phel93Ser Asnlβ3Gln 4 Alal68Asp + Asnl7OAsp 4 Ser207Glu 4

Thr217Gly 4 Ser220Asp Gly 67Asp 4 AsnlOβGlu 4 Thrll3Ser 4 Glyl36Gln 4

AsnlββGlu 4 Leu221Cys Asn ββGln 4 GlyllOAsp 4 Alall4His 4 Glyl36Glu 4

Thrl37Asp 4 Ser222Glu Gly 67Asp 4 Asn βδSer 4 Gly 72Pro 4 Glyl39Ser 4

Thrl67Glu 4 Thrl95Asp Asn ββAsp 4 Asn βδGln 4 Serl91Glu 4 Ser207Asp 4

Trp20δVal 4 Ser220Asp Serl09Glu + Alall4Glu + Leul34Ile 4 Glyl36Ser 4

Ala219Glu 4 Ser220Asp ValllSGlu + AlalβδHis + Prol69Asn 4 Tyr210Asp 4

Ser216Glu 4 Ala219Asp Asn ββGlu + AsnlOβAsp + Asnl70Ser 4 Trp20δThr 4

Ser21βAsp Tyr21δGly Glyl62Asn 4 Asnl7OAsp 4 Phel93Glu 4 Tyr210Gln 4

Ser220Glu 4 Thr224Asp GlylOδSer 4 Leul34Pro 4 Thrl37Asp 4 Prol69Glu 4

Ser207Glu 4 Thr217Gly Thrl37Asp 4 Vall38Glu 4 Asnl63Glu 4 Alalβ4Asn 4

AlalβδSer 4 Thr217Glu Glyl62Glu + Trp208Glu Tyr210Val 4 Thr217Glu

Ser220Glu 4 Gly223Pro Glyl35Asp + Vall38Thr + Ser207Asp + Thr217Pro 4

Tyr21δlle 4 Ser220Glu Leul04Gly 4 Serl92Asp 4 Thrl95Asp + Thr217Pro 4

Ser220Glu 4 Leu221Asn Asn ββSer + Glyl36Glu 4 Vall3δGlu + Trp20δLeu +

Thr217Asp 4 Ser220Glu Gly 69Ser + Vall03Asp 4 Serl09Asp 4 Serl91Asp 4

Gly20βGln Tyr210Pro Alall4Gly 4 Glyl62Asp + Pro214Gly + Thr215Asp 4

Thr217Glu 4 Ser220Glu Thrl37Gln 4 Glyl39Asp Glyl65Pro + Tyrl71Glu +

Thr217Glu 4 Ser220Glu Gly 67Glu + Aspl05Glu + Asnl40Glu + Phel93Gly +

Trp20δLeu 4 Leu221Cys Leul04Glu Trpll2Leu 4 Phel93Leu + Ser207Asp 4

Trp208Ala 4 Thr224Asp Ser 70Giu 4 Thrll3Gly Glyl62Ser 4 Asnl63Asp 4

Alal64Glu 4 Ser220Asp Gly 69Giu 4 Ser 70Asp 4 τhr:::Giu 4 Ser220Asp 4

Leu221Pro 4 Gly223Ser Asn ββGln 4 Leul04Pro 4 Glyl36Glu 4 Asnl63Asp 4

Tyrl71Ile 4 Leu221Glu Ser 7OAsp + Leul04Glu 4 AlalβδAsn 4 Prol69Glu 4

Thrl95Asp 4 Trp20δPhe Ser 70Glu 4 Glyl62Glu 4 Trp20δGlu 4 Thr217Gly 4

Leu221Thr 4 Ser222Asp Gly 69Asn 4 Thrl37Glu + Glyl39Pro 4 AsnlββAsp 4

Prol69Asp 4 Trp208Asp Gly 69Glu 4 Leul04Glu 4 Valll5Ser 4 Asnl40Ser 4

Serl92Glu 4 Pro214Asp Leul04His 4 Aspl05Glu 4 Vall3δHis 4 Asnl63Asp 4

Serl92Glu 4 Thr217Asn Alall4Asn 4 Asnl63Giu 4 Asnl70Ser 4 Serl92Asp 4

Ala204Asn 4 Thr217Asp Asn βδGln 4 Thr 73Asp 4 Serl09Glu 4 Trp20δPhe 4

Pro214Glu 4 Ser220Asp Valll5Cys 4 Leul34Asp 4 Glyl35Pro 4 Alal64Thr 4

Asnl7OAsp 4 Trp208Asp Asn ββGlu Gly 69Glu 4 Trpll2Gln 4 Glyl35Asn 4

Glyl39Asp 4 Asnl70Glu Thr 73Gly + Serl07Glu 4 Glyl35Asp 4 Prol69Asp 4

Trp20δThr 4 Leu221Asn Gly 67Ser 4 Ser 70Glu 4 Glyl36Asp 4 Prol69Asn 4

Leu221Asp 4 Gly223Asp Ser 7OAsp 4 GlyllOGlu 4 Vall3δSer 4 Tyr210Val +

Leu221Asp 4 Gly223Asp Asn ββAsp 4 Leul04Glu 4 Serl09Glu 4 Tyr210Asn 4

Thr217Asn 4 Ser220Asp Asn ββGlu + Gly 67Asp 4 Asnl63Glu 4 Ser207Glu 4

Thr215Gly 4 Leu221Thr Gly 69Glu + Tyr210Glu 4 Thr215Asp 4 Thr217Gln 4

Tyr21δGln 4 Ser220Glu Ser 7OAsp 4 Thrl37Glu 4 Alal64His 4 Thrl67Gly +

Trp2OδAsp 4 Ser220Glu Vall3δMet 4 Glyl39Gln 4 Asnl40Glu 4 AlalβδPro +

Tyrl71Glu 4 Thr215Glu Vall03Gly AsnlOβSer 4 Serl07Asp 4 ThrlllAsp 4

Ser222Glu 4 Gly223Glu Gly 72Gln + AsnlOβGlu 4 GlyllOAsp 4 Thrll3Glu 4

Glyl35Asn 4 Ala219Glu Serl09Glu + Thrll3Asp 4 Trp20δlle 4 Ser220Glu 4

Leu221Asp 4 Thr224Ser Gly 67Asn 4 Ser 70Asp 4 GlylOδAsp 4 Asnl70Glu 4

Thr215Pro 4 Leu221Thr Gly β7Asp 4 Serl07Asp 4 Thrl67Gly 4 Tyr210Asn 4

Ser220Asp 4 Ser222Glu Gly 69Asp + Thr 73Glu 4 AsnlOβGln 4 ThrlllAsn 4 Vall38Glu 4 Thr217Asp ThrlllAsn + Serl91Asp + Thrl95Asp + Ala204Thr +

Ala219Ser 4 Ser220Glu Serl09Asp 4 Serl91Glu + Tyr210Val + Thr215Asn 4

Leu221Gly 4 Ser222Glu Thr 73Glu 4 Leul04Asp 4 AsnlOβGln 4 Thrl95Ser 4

Ser207Glu 4 Ser220Asp Asn ββAsp 4 Thr 73Asp 4 Leul04Glu 4 Glyl36Gln 4

Glyl62Asp 4 Leu221Gly Gly 72Asn 4 Leul04Asp 4 Asnl63Asp 4 Tyrl71Ser 4

Serl91Glu 4 Thr217Asn Valll5Asp 4 Glyl36Gln 4 Tyr210Glu 4 Tyr21δCys

Leu221Asn 4 Ser222Asp Thr 73Asp 4 Asnl40Ser 4 Asnl63Glu 4 Thrl95Asp 4

Ser220Asp 4 Leu221Pro Asn 68Glu 4 Leul04Asp 4 Prol69Asn 4 Trp206Val 4

Thr217Asn 4 Gly223Glu GlyllOGlu Thrl67Gln 4 Serl92Glu 4 Ser207Glu 4

Trp206Cys 4 Ser220Asp Vall03Gly 4 Glyl3βGlu 4 Asnl OAsp 4 Ser207Asp

Tyr213Asn 4 Ser220Asp Thrll3Asp + Glyl36Asp 4 Thri37Gln 4 Asnl63Glu +

AsnlββGlu 4 Trp208Phe Asn ββGlu 4 Gly 69Glu 4 Serl09Asp 4 Serl92Asp 4

Phel93Val 4 Tyr218Ala Ser 70Glu 4 Serl41Asp 4 Asnl63Ser 4 Trp20δMet +

Tyr210Pro 4 Thr215Asp Aspl05Glu 4 GlyllOAsn + ThrlllGlu 4 Leul34Ala +

Serl41Glu 4 Phei93Tyr Asn ββGlu 4 Gly 69Ser 4 GlylOδAsp 4 ThrlllAsp

Thrl95Gly 4 Tyr210Leu Glyl65Glu 4 Tyrl71Asn 4 Ser207Glu 4 Thr215Glu 4

Thr217Gly + Thr224Glu Asn ββGln 4 Thr 73Ser 4 Glyl39Asn 4 Pro214Asp 4

Ser220Asp 4 Leu221Ile AsnlββGlu 4 Serl92Asp 4 Trp20δThr 4 Tyr210Gly 4

Thr215Asp 4 Ser222Glu Asn ββAsp 4 Serl91Asp 4 Thr215Asn 4 Ala219Asp +

Ser222Asp 4 Gly223Gln Aspl05Glu + GlylOδGlu 4 Thrl95Asn 4 Ile209Leu 4

Pro214Asp 4 Ser220Asp ThrlllGlu + Val13δAsp + Asnl63Ser 4 Tyr218Met +

Leu221Glu Gly223Asp Gly 67Glu + Vall3δGlu Serl41Glu 4 Trp208Ile +

Pro214Asn 4 Thr215Asp Trpll2Leu + Thrll3Gln 4 Glyl65Glu 4 Thrl67Asp

Thr217Glu 4 Ser220Asp Gly 67Gln 4 Asn βδAsp 4 Gly 69Asp 4 Phel93Ala

Ser216Glu 4 Ser222Glu Ser 70Asp + Valll5Glu 4 Glyl62Ser 4 AlalβδThr 4

Tyr210Gly 4 Thr217Asp Ser 7OAsp 4 Glyl36Asn 4 Glvl65Glu 4 Tyr210Asn

Thr217Asp 4 Leu221His Thr 73Ser 4 Vall03Asn 4 Serl09Asp + Serl92Asp 4

Ser207Asp 4 Trp208Ile AsnlOβAsp 4 Serl92Glu 4 Phel93Pro + Ser207Glu 4

Thr217Gln 4 Tyr21δlle Serl41Asp -+- Thrl67Asp 4 AlalβδAsp + Serl91Asp 4

Aia219Asn 4 Leu221Thr Asn ββGln 4 Serl41Asp 4 Asnl70Ser + Thr217Gln 4

Ser220Asp 4 Gly223Asp Thr 73Gly 4 Aspl05Glu 4 Alall4His + Ser220Glu 4

Leu221Asn 4 Gly223Glu AsnlOβAsp 4 Asnl40Gln 4 Trp20δSer 4 Tyr210Cys 4

Ser220Asp 4 Gly223Glu Gly 67Gln 4 Aspl05Glu 4 Alal64Asp 4 Prol69Asn 4

Tyr218Leu 4 Gly223Asp Trpll2Val 4 AsnlββAsp 4 Phel93Gly 4 Pro214Glu 4

Ser220Asp 4 Ser222Glu GlylOδAsp 4 ThrlllAsn 4 Serl92Glu 4 Thr217Asn 4

Ala219Glu 4 Ser220Asp Vall03Ser 4 Valll5Asp 4 Glyl39Glu + AsnlββSer 4

Ser207Glu 4 Ser220Glu Leul04Asp 4 Aspl05Glu 4 Asnl40Glu 4 Thrl67Asp 4

Pro214Gly 4 Ala219Gly Gly 67Asp 4 Asn βδAsp 4 ThrlllAsp 4 Alalβ4Gln 4

Phel93Ser 4 Ser207Asp Serl09Glu 4 Trp20δAsp 4 Ser216Glu + Thr217Asp 4

Tyr21δAsn 4 Leu221His Serl41Asp 4 AlalβδGlu 4 Tyr210Met 4 Ser216Glu 4

Thr217Asp 4 Tyr21δAla Serl09Asp 4 GlyllOAsp 4 AsnlββAsp 4 Asnl70Ser 4

Thr215Ser 4 Leu221Asp GlyllOGlu 4 Thrl67Glu 4 AlalβδAsp + Trp206Met 4

Ser220Asp 4 Thr224Gln Aspl05Glu 4 AsnlOβAsp 4 Glyl65Asp + Thrl95Ser 4

Tyr216Ile 4 Ser220Asp AsnlOβGlu 4 Serl41Asp 4 Tyr218Ser 4 Ala219Thr 4

Ser220Asp 4 Leu221Glu Thr 73Gln 4 ThrlllGlu 4 AsnlββAsp 4 Trp206Ser 4

Ser220Asp 4 Leu221Asp Serl07Glu 4 AlalβδAsn 4 Prol69Asn 4 Serl91Asp 4

Serl92Asp 4 Thr217Asp Alall4Glu AsnlββSer 4 Thr212Pro 4 Thr217Glu 4

Tyr216Asp 4 Thr224Asp AsnlOβGlu 4 Serl07Asp 4 Glyl39Asp 4 Ala203Asn 4

Thr217Ser 4 Ser220Asp Thrll3Asn 4 Glyl39Ser 4 Serl41Glu + Thr215Glu 4

Ser216Asp 4 Ser220Asp Gly 69Gln 4 Asnl40Glu 4 Glyl62Glu + Asnl63Asp 4

Tyr210Gln 4 Ser220Asp Asn 68Asp 4 Alall4Pro 4 Glyl35Glu + Trp2OδAsp

Leu221Ile 4 Gly223Asp Asn 68Asp 4 Alal64Glu 4 Trp208Cys 4 Tyr210Glu +

Ala219Thr 4 Ser220Asp ThrlllAsp + AsnlββAsp 4 Asnl70Gln + Tyr210Asp + Thr212Ser 4 Ser220Glu Serl41Glu 4 Glyl65Asp + Trp20δlle 4 Tyr210Asp +

Thr217Gln 4 Ser220Asp Asn ββGln + Vall03Asp + GlylOδPro 4 Thrl37Glu 4

Tyr210Glu 4 Ser220Glu GlylOδGlu 4 Thrl37Gln 4 Asnl63Glu 4 Tyr210Glu +

Ser220Asp 4 Leu221Cys Gly β9Gln 4 Ser 70Glu 4 Glyl35Glu 4 Asnl63Asp 4

Thr217Gly 4 Ser220Asp AsnlOβGlu 4 Asnl40Glu 4 Asnl63Glu 4 Asnl70Glu 4

Thrl95Asn 4 Leu221His AsnlOβGln 4 AsnlββAsp 4 AlalβδAsn 4 Asnl70Glu 4

Ser207Glu 4 Ser220Asp Asn ββAsp 4 Ser 70Asp 4 GlyllOGln + Glyl36Asp 4

Pro214Gln + Ser220Asp Asn βδSer 4 Prolβ9Asp + Serl92Glu + Ala219Asp 4

Gly223Ser 4 Thr224Asp Thr 73Pro + Glyl35Ser 4 Phel93Glu + Gly206Gln 4

Tyr210Asp 4 Thr217Glu Trpll2Glu 4 Valll5Gln 4 Asnl70Gln + Thr217Asp 4

Tyr216Met 4 Ser220Asp GlyllOAsn 4 Valll5Glu + Thrl37Gln 4 Ile209Gly 4

Thr217Asp 4 Ser220Glu Glyl08Glu + Thrll3Gln 4 Pro214Asn 4 Thr217Glu 4

Tyr218Gln 4 Ser220Asp GlyllOSer 4 ThrlllGly Thrll3Glu 4 Phel93Pro 4

Thr217Glu 4 Ser220Asp AsnlββGlu 4 Trp20δlle 4 Tyr210Met Thr217Glu 4

Tyr21δVal 4 Ser220Asp Ser 70Glu + Vall03Glu 4 Valll5Gln 4 Prolβ9Ser 4

Tyr210Ala 4 Ser220Asp Asn ββAsp + Serl07Asp 4 Trp20δMet 4 Tyr210Pro 4

Ser220Asp 4 Ser222Glu GlyllOSer + ThrlllAsp + Glyl35Gln 4 Glyl36Asp +

Thrl37Asn Trp208Glu Thrl67Asp + AlalβδSer 4 Prol69Glu + Trp208Thr 4

Thr215Asp + Leu221Asp Val-115Gln AlalβδGln Prol69Gln Phel93Asp ^*Tyr210Ser Leu221Asp Asn ββGlu + Glyl36Pro + Asnl40Gln + Serl91Asp 4

Trp208Asp 4 Leu221His Glyl36Glu 4 Glyl39Glu + Serl92Asp 4 Ala203Asn 4

Trp2OδAsp 4 Leu221His Gly 67Asn 4 Thrll3Gln 4 Thrl37Glu + Thrlβ7Asp 4

Ala219Gln 4 Leu221Glu Gly 69Asn 4 Thr 73Asp + ThrlllPro + AsnlββAsp

Serl91Asp + Leu221Val Vall38Pro + AsnlββGlu + Serl91Asp + Tyr218Asn

Ser220Asp + Leu221Gln Aspl05Glu + Glyl39Asn 4 Glyl65Glu + Trp208Met +

Tyr21δThr + Gly223Asp GlyllOAsn 4 ThrlllPro + Alall4Asp + Asnl7OAsp +

Trp206Asp 4 Tyr210Asp Gly 67Gln 4 Gly 69Glu + Serl07Asp 4 Thrl67Glu 4

AlalβδAsn 4 Ser207Asp Gly 69Glu 4 GlylβSGlu + Trp206Cys 4 Tyr210Cys 4

Pro214Asp 4 Ser220Asp Ser 70Glu 4 Gly 72Gln 4 Vall03Thr 4 Serl09Glu 4

Thrll3Glu 4 Ser220Asp Gly 69Pro 4 Valll5Glu 4 Tyr210Asn 4 Ser216Asp 4

Ser220Glu 4 Ser222Glu Glyl36Glu 4 Trp20δSer 4 Ser216Asp 4 Ser220Asp

Leu221Asn + Ser222Asp Leul04Glu 4 Vaill5Pro Glyl62Ser 4 AsnlββAsp 4

Ser220Asp 4 Ser222Asp Gly 67Glu 4 Vall38Asp + Thr217Ser 4 Ser220Asp 4

Leu221His 4 Ser222Asp Asn βδAsp 4 Asnl70Glu 4 Gly206Asn 4 Ser207Asp 4

Trp20δlle 4 Leu221Asp AsnlOβGln 4 Serl09Glu + Thrll3Asp 4 Leul34Gln 4

Thrl37Glu 4 Trp206Asn AsnlOβGlu 4 Glyl39Gln Thrl67Glu 4 Prol69Gln 4

Serl91Asp 4 Ser207Glu Serl07Glu 4 Valll5Asp + Glyl65Asn 4 Trp20δThr 4

Tyr218Asp 4 Ser220Glu Gly 72Asn 4 AsnlOβGln 4 Glyl39Asp 4 Prol69Glu 4

Trp208Pro 4 Ser220Glu Vall36Asp + Serl41Glu + Prol69Ser 4 Ser207Glu 4

Ala219Glu 4 Leu221Thr AsnlOβGln 4 Alall4Asp + Glyl35Asp 4 Thrl95Asn 4

Ser207Glu 4 Ser220Asp Ser 70Asp 4 AsnlββGlu 4 Asnl70Gln 4 Ser216Glu 4

Thr217Gln 4 Thr224Gly Gly 69Asp 4 Asnl40Ser 4 Glyl62Pro 4 Trp208Gly 4

Thr217Glu 4 Leu221Asp Asn βδAsp 4 Serl07Asp 4 Alall4Gln 4 Glyl62Asp 4

Thr217Pro 4 Ser220Glu Ser 70Asp 4 ThrlllAsp + Ile209Met 4 Thr217Gly 4

Ser220Glu 4 Leu221Asn Ser 70Asp Leul04His + ThrlllGln 4 Tyrl71Ser 4

_Phel93Asp Ser220Glu Thrll3Gly 4 Glyl39Pro 4 Serl41Glu 4 Tyrl71Glu 4

Serl92Asp 4 Ile209Ser Trpll2Ala + Thrll3Glu 4 Vall3δGln 4 Serl41Asp +

Ser216Asp 4 Leu221Ser Gly 69Asn 4 AsnlOβAsp 4 Trpll2Gly 4 Glyl62Asp +

Phel93Glu 4 Ser220Glu Asn β8Ser + Vall03Glu + AsnlOβAsp 4 AlalβδPro +

Prol69Asp 4 Pro214Glu Leul04Glu 4 Alall4Ser 4 Asnl40Ser 4 Glyl65Asp 4

Ser207Asp 4 Ser220Glu Asn βδAsp 4 Serl09Glu 4 Glyl39Glu 4 Ala204Ser 4

Leu221His 4 Gly223Asn ThrlllGly + Vall36Pro 4 Glyl39Asp 4 Ser207Glu 4

Ser216Glu 4 Ser220Asp AsnlOβAsp 4 Asnl70Ser 4 Serl92Glu 4 Thrl95Asp + Trp208His 4 Ser220Glu AsnlO^βAsp + Glyl62Asp + AsnlββGln + Thrl67Asp 4

Pro214Asp 4 Aia219Thr ValllSGlu 4 Glyl36Glu + Ser207Glu 4 Tyr218Gly +

Ala219Pro Leu221Pro Glyl36Ser 4 Glyl62Asp + Asnl63Gln 4 Alal64His 4

Ser216Asp 4 Ser222Glu Asn 68Ser 4 Thr 73Glu 4 AsnlββGlu 4 Asnl70Gln 4

Tyrl71Gly 4 Leu221Asp Thrll3Glu 4 Valll5Cys 4 Thrl37Glu 4 Serl91Glu 4

Thr217Ser 4 Thr224Glu Gly 67Asp 4 Serl07Asp 4 Glyl39Asp + Glyl65Pro 4

Thrl67Ser 4 Thr217Glu Thr 73Gly + GlylOδAsp + Thrl37Glu + Glyl62Glu +

Tyr210Gln 4 Ser220Asp Leul04Asp 4 Glyl39Pro 4 Tyrl71Asp + Serl91Asp 4

Ser222Glu 4 Thr224Ser Thrll3A_sp 4 Prol69Glu 4 Tyr210Gln 4 Pro214Ser 4

Ser220Glu 4 Gly223Asp GlylOδAsp 4 Glyl3βGlu 4 Serl41Asp 4 Thrl95Asp 4

Thr215Ser 4 Thr217Gln Serl07Asp 4 Asnl63Glu + Prol69Gly 4 Ser207Asp 4

Trp206Asn 4 Gly223Asn GlyllOAsp 4 Asnlβ3Asp + Serl91Asp 4 Thrl95Gln 4

Tyr210Glu 4 Thr217Pro Vall03Glu 4 GlylOδAsp + Ala203Gly 4 Trp208Met 4

Ala219Asp 4 Thr224Glu Thr 73Gln 4 ThrlllAsp 4 Leul34Asp 4 Glyl36Ser 4

Asnl63Gln 4 Ser207Asp

π. Cleaning Compositions

In another embodiment of the present invention, an effective amount of one or more of the enzyme variants are included in compositions useful for cleaning a variety of surfaces in need of proteinaceous stain removal. Such cleaning compositions include detergent compositions for cleaning hard surfaces, unlimited in form (e.g., liquid and granular); detergent compositions for cleaning fabrics, unlimited in form (e.g., granular, liquid and bar formulations); dishwashing compositions (unlimited in form); oral cleaning compositions, unlimited in form (e.g., dentifrice, toothpaste and mouthwash formulations); denture cleaning compositions, unlimited in form (e.g., liquid, tablet); and contact lens cleaning compositions, unlimited in form (e.g., liquid, tablet).

The cleaning compositions also comprise, in addition to the Thermitase variants described hereinbefore, one or more cleaning composition materials compatible with the protease enzyme, the term "cleaning composition material", as used herein, means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e g , liquid, granule, bar, spray, stick, paste, gel), which materials are also compatible with the Thermitase variant used in the composition the specific selection of cleaning composition materials are readily made by considering the surface material to be cleaned, the desired form of the composition for the cleaning condition during use (e g , through the wash detergent use) The term "compatible", as used herein, means the cleaning composition materials do not reduce the proteolytic activity of the Thermitase variant to such an extent that the protease is not effective as desired during normal use situations Specific cleaning composition materials are exemplified in detail hereinafter

As used herein, "effective amount of enzyme variant" refers to the quantity of enzyme variant necessary to achieve the enzymatic activity necessary in the specific cleaning composition Such effective amounts are readily ascertained by one of ordinary skill in the art and is based on many factors, such as the particular enzyme variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e g , granular, bar) composition is required, and the like Preferably the cleaning compositions comprise from about 0 0001 % to about 10% of one or more enzyme variants of the present invention, more preferably from about 0 001 % to about 1 %, more preferably still from about 0 01 % to about 0 1 % Several examples of various cleaning compositions wherein the enzyme variants may be employed are discussed in further detail below All parts, percentages and ratios used herein are by weight unless otherwise specified

As used herein, "non-fabric cleaning compositions" include hard surface cleaning compositions, dishwashing compositions, oral cleaning compositions, denture cleaning compositions and contact lens cleaning compositions A Cleaning Compositions for Hard Surfaces, Dishes and Fabrics

The enzyme variants of the present invention can be used in a variety of detergent compositions where high sudsing and good insoluble substrate removal are desired Thus the enzyme variants can be used with various conventional ingredients to provide fully-formulated hard-surface cleaners, dishwashing compositions, fabric laundering compositions and the like Such compositions can be in the form of liquids, granules, bars and the like Such compositions can be formulated as modern "concentrated" detergents which contain as much as 30%-60% by weight of surfactants

The cleaning compositions herein can optionally, and preferably, contain various anionic, nonionic, zwittenonic, etc , surfactants Such surfactants are typically present at levels of from about 5% to about 35% of the compositions

Nonlimiting examples of surfactants useful herein include the conventional C-| i-C-] 8 alkyl benzene sulfonates and primary and random alkyl sulfates, the Cιo-C-|8 secondary (2,3) alkyl sulfates of the formulas CH₃(CH₂)x(CHOSO3)-M⁺)CH₃ and CH₃(CH₂)y(CHOSO3-M⁺) CH₂CH₃ wherein x and (y+1 ) are integers of at least about 7, preferably at least about 9, and M is a water-solubilizmg cation, especially sodium, the C-I Q- C-| 8 alkyl alkoxy sulfates (especially EO 1-5 ethoxy sulfates), C-|o-C*i 8 alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the C-|n- C-j s alkyl polyglycosides, and their corresponding sulfated polyglycosides, C-]2-C*| 8 alpha-suifonated fatty acid esters C12-C-.8 ^a'ky' and alkyl phenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy), C-12-C18 betaines and sulfobetames ("sultames"), C^ Q-C^ Q amine oxides, and the like The alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein (Use of such surfactants in combination with the aforesaid amine oxide and/or betaine or sultame surfactants is also preferred, depending on the desires of the formulator ) Other conventional useful surfactants are listed in standard texts Particularly useful surfactants include the C*ιo-C*i8 N-methyl glucamides disclosed in US Patent 5, 194,639, Connor et al , issued March 16, 1993, incorporated herein by reference

A wide variety of other ingredients useful in detergent cleaning compositions can be included in the compositions herein, including other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, etc If an additional increment of sudsing is desired, suds boosters such as the C*|o-C-|6 alkolamides can be incorporated into the compositions, typically at about 1% to about 10% levels The C10-C14 monoethanol and diethanol amides illustrate a typical class of such suds boosters Use of such suds boosters with high sudsing adjunct surfactants such as the amine oxides, betaines and sultames noted above is also advantageous If desired, soluble magnesium salts such as MgCl2, MgSO4, and the like, can be added at levels of, typically, from about 0 1 % to about 2%, to provide additionally sudsing

The liquid detergent compositions herein can contain water and other solvents as carriers Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol are suitable Monohydπc alcohols are preferred for solubiliz g surfactants, but polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e g , 1 ,3-propanedιol, ethylene glycol, glycerine, and 1 ,2-propanedιol) can also be used The compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of such carriers

The detergent compositions herein will preferably be formulated such that during use in aqueous cleaning operations, the wash water will have a pH between about 6 8 and about 11 0 Finished products thus are typically formulated at this range Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc , and are well known to those skilled in the art

When formulating the hard surface cleaning compositions and fabric cleaning compositions of the present invention, the formulator may wish to employ various builders at levels from about 5% to about 50% by weight Typical builders include the 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccmates, layered silicates, phosphates, and the like Other conventional builders are listed in standard formularies

Likewise, the formulator may wish to employ various additional enzymes, such as cellulases, pases, amylases and proteases in such compositions, typically at levels of from about 0.001 % to about 1 % by weight Various detersive and fabric care enzymes are well-known in the laundry detergent art

Various bleaching compounds, such as the percarbonates, perborates and the like, can be used in such compositions, typically at levels from about 1 % to about 15% by weight If desired, such compositions can also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art Usage levels typically range from about 1 % to about 10% by weight

Various soil release agents, especially of the anionic oligoester type, various chelat g agents, especially the ammophosphonates and ethylenediaminedisuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamme, various dispersing agents, especially polyacrylates and polyasparatates, various brighteners, especially anionic brighteners, various suds suppressors, especially silicones and secondary alcohols, various fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1 % to about 35% by weight. Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials.

Enzyme stabilizers may also be used in the cleaning compositions. Such enzyme stabilizers include propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1 % to about 1 %) and calcium formate (preferably from about 0.1 % to about 1 %). 1. Hard surface cleaning compositions

As used herein "hard surface cleaning composition" refers to liquid and granular detergent compositions for cleaning hard surfaces such as floors, walls, bathroom tile, and the like. Hard surface cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001 % to about 10%, more preferably from about .01 % to about 5%, more preferably still from about .05% to about 1 % by weight of active enzyme of the composition. In addition to comprising one or more of the enzyme variants, such hard surface cleaning compositions typically comprise a surfactant and a water-soluble sequestering builder. In certain specialized products such as spray window cleaners, however, the surfactants are sometimes not used since they may produce a filmy/streaky residue on the glass surface.

The surfactant component, when present, may comprise as little as 0.1 % of the compositions herein, but typically the compositions will contain from about 0.25% to about 10%, more preferably from about 1 % to about 5% of surfactant.

Typically the compositions will contain from about 0.5% to about 50% of a detergency builder, preferably from about 1% to about 10%.

Preferably the pH should be in the range of about 8 to 12. Conventional pH adjustment agents such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used if adjustment is necessary.

Solvents may be included in the compositions. Useful solvents include, but are not limited to, glycol ethers such as diethyleneglycol monohexyl ether, diethyleneglycol monobutyl ether, ethyieneglycol monobutyl ether, ethyieneglycol monohexyl ether, propyleneglycol monobutyl ether, dipropyleneglycol monobutyl ether, and diols such as 2,2,4-trιmethyl-1 ,3-pentanedιol and 2-ethyl-1 ,3-hexanedιol When used, such solvents are typically present at levels of from about 0 5% to about 15%, preferably from about 3% to about 11 %

Additionally, highly volatile solvents such as isopropanol or ethanol can be used in the present compositions to facilitate faster evaporation of the composition from surfaces when the surface is not rinsed after "full strength" application of the composition to the surface When used, volatile solvents are typically present at levels of from about 2% to about 12% in the compositions

The hard surface cleaning composition embodiment of the present invention is illustrated by the following examples Examples 7-12

Liquid Hard Surface Cleaning Compositions

Example No

Component 9 10 11 12 0 50 0 02 0 03 0 10 0 03 0 20 0 02

0 50 0 50 - 0 50

1 30 - 1 30 6 30 6 30 6 30 6 30 6 30

Water^* balance to 100%

*Dιsodιum N-dιethyleneglycol-N,N-ιmιnodιacetate **Na4 ethylenediamine diacetic acid ***Dιethyleneglycol monohexyl ether ^****AII formulas adjusted to pH 7

In Examples 7-10, the Thermitase variants recited in Tables 2-36, among others, are substituted for Gln66Asn, with substantially similar results.

In Examples 11 -12, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Gln66Asn and Gly206Asn, with substantially similar results.

Examples 13-18 Spray Compositions for Cleaning Hard Surfaces and Removing Household Mildew

Product pH is about 7.

In Examples 13-16, the Thermitase variants recited in Tables 2-36, among others, are substituted for Thr111Asn, with substantially similar results.

In Examples 17-18, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Thr111Asn and Thr217Gly + Ser222Glu, with substantially similar results. 2. Dishwashing Compositions

In another embodiment of the present invention, dishwashing compositions comprise one or more enzyme variants of the present invention. As used herein, "dishwashing composition" refers to all forms for compositions for cleaning dishes, including but not limited to, granular and liquid forms. The dishwashing composition embodiment of the present invention is illustrated by the following examples. Examples 19-24 Dishwashing Composition

Component 19

Gly139Asn 0 05

Ser207Glu + Tyr210Ser + Gly223Asn

C-12-C-.4 N-methyl- glucamide 0 90

Ci 2 ethoxy (1 ) sulfate 12 00

2-methyl undecanoic acid 4 50

C*]2 ethoxy (2) carboxylate 4 50

C<|2 alcohol ethoxylate (4) 3 00

C-] 2 amine oxide 3 00

Sodium cumene sulfonate 2 00

Ethanol 4 00

Mg⁺⁺ (as MgCl2) 0 20

Ca⁺⁺ (as CaCl2) 0 40

Water

Product pH is adjusted to 7

In Examples 19-22, the Thermitase variants recited in Tables 2-36, among others, are substituted for Gly139Asn, with substantially similar results

In Examples 23-24, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Gly139Asn and Ser207Glu + Tyr210Ser + Gly223Asn, with substantially similar results 3 . Fabric cleaning compositions

In another embodiment of the present invention, fabric cleaning compositions comprise one or more enzyme variants of the present invention As used herein, "fabric cleaning composition" refers to all forms for detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms Preferred fabric cleaning compositions are those in the liquid form a Granular fabric cleaning compositions

The granular fabric cleaning compositions of the present invention contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0 001 % to about 10%, more preferably from about 0.005% to about 5%, more preferably from about 0.01 % to about 1 % by weight of active enzyme of the composition. In addition to one or more enzyme variants, the granular fabric cleaning compositions typically comprise at least one surfactant, one or more builders, and, in some cases, a bleaching agent.

The granular fabric cleaning composition embodiment of the present invention is illustrated by the following examples.

Examples 25-28 Granular Fabric Cleaning Composition

In Examples 25-26, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ala168Asn, with substantially similar results.

In Examples 27-28, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Ala168Asn and Tyr210Gly + Thr212Pro + Pro214Gly + Ser222Asp, with substantially similar results. Examples 29-32 Granular Fabric Cleaning Composition

In Examples 29-30, the Thermitase variants recited in Tables 2-36, among others, are substituted for Thr195Pro, with substantially similar results

In Examples 31-32 any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Thr195Pro and Trp208Phe + Tyr213Met + Thr215Pro + Ser220Glu + Thr224Gly, with substantially similar results

Examples 33-36 Granular Fabric Cleaning Composition

In Examples 33-34, the Thermitase variants recited in Tables 2-36, among others, are substituted for Gly67Ser + Gly72Ser, with substantially similar results

In Examples 35-36, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Gly67Ser + Gly72Ser and Tyr171Thr with substantially similar results

Examples 37-40 Granular Fabric Cleaning Composition

Example No

Component 37 38 39 40

In Examples 37-38, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser192Asp + Phe193Leu + Thr195Asn, with substantially similar results

In Examples 39-40, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser192Asp + Phe193Leu + Thr195Asn and Tyr213Ser + Thr217Gly + Gly223Glu, with substantially similar results

Examples 41-42 Granular Fabric Cleaning Composition

Examples 43-44 Granular Fabric Cleaning Composition

Example No.

Component 43 44_

Sodium linear C-J2 alkyl benzene-sulfonate

Sodium sulfate

Zeolite A

Sodium nitrilot acetate

Polyvinyl pyrrolidone

Tetraacetylethylene diamine

Boric acid

Perborate

Phenol sulphonate

Pro205Gly + Trp208Leu + Thr217Gln

+ Leu221 Gln + Gly223Gln + Thr224Glu

Fillers (e.g., silicates; carbonates; Up to 100 Up to 100 perfumes; water)

Example 45 Compact Granular Fabric Cleaning Composition

Component Weight %

Alkyl Sulphate 8.0

Alkyl Ethoxy Sulphate 2.0

Mixture of C25 and C45 alcohol 3 and 7 times ethoxylated 6.0

Polyhydroxy fatty acid amide 2.5

Zeolite 17.0

Layered silicate/citrate 16.0

Carbonate 7.0

Maleic acid acrylic acid copolymer 5.0

Soil release polymer 0.4

Carboxymethyl cellulose 0.4

Poly (4-vinylpyridine) -N-oxide 0.1

Copolymer of vinylimidazole and vinylpyrrolidone 0.1

PEG2000 0.2

Asn66Ser + Ser70Glu + Gly72Asn 0.5

Lipase 0.2

Cellulase 0.2

Tetracetylethylene diamine 6.0 Percarbonate

Ethylene diamine disuccmic acid

Suds suppressor

Dιsodιum-4 4'-bιs (2-morpholιno -4-anιlιno-s-trιazιn-6- ylamino) stιlbene-2,2'-dιsulphonate

Dιsodιum-4,4'-bιs (2-sulfostyπl) biphenyl

Water, Perfume and Minors

Example 46 Granular Fabric Cleaning Composition

Component Weight %

Linear alkyl benzene sulphonate 7 6

C-I 6-C-I 8 alkyl sulfate 1 3

^14-15 alcohol 7 times ethoxylated 4 0

Coco-alkyl-dimethyl hydroxyethyl ammonium chloride 1 4

Dispersant 0.07

Silicone fluid 0 8

Tπsodium citrate 5.0

Zeolite 4A 15.0

Maleic acid acrylic acid copolymer 4 0

Diethylene triamme penta methylene phosphonic acid 0 4

Perborate 15 0

Tetraacetylethylene diamine 5.0

Smectite clay 10.0

Poly (oxy ethylene) (MW 300,000) 0.3

Ser109Glu + Thr113Gly 0 4

Lipase 0.2

Aηnylase 0 3

Cellulase 0.2

Sodium silicate 3.0

Sodium carbonate 10.0

Carboxymethyl cellulose 0.2

Brighteners 0.2

Water, perfume and minors Up to 100 Example 47 Granular Fabric Cleaning Composition

Component Weight %

Linear alkyl benzene sulfonate 6 92

Tallow alkyl sulfate 2 05

C-|4-| 5 alcohol 7 times ethoxylated 4 4

C-J2-15 alkyl ethoxy sulfate - 3 times ethoxylated 0 16

Zeolite 20 2

Citrate 5 5

Carbonate 15 4

Silicate 3 0

Maleic acid acrylic acid copolymer 4 0

Carboxymethyl cellulase 0 31

Soil release polymer 0 30

Glyl 35Gln + VaM 38Asp + Glyl 39Ser 0 2

Lipase 0 36

Cellulase 0 13

Perborate tetrahydrate 11 64

Perborate monohydrate 8 7

Tetraacetylethylene diamine 5 0

Diethylene tramme penta methyl phosphonic acid 0 38

Magnesium sulfate 040

Bnghtener 0 19

Perfume, silicone, suds suppressors 0 85

Minors Up to 100 b. Liquid fabric cleaning compositions

Liquid fabric cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0 005% to about 5%, more preferably from about 0 01 % to about 1 %, by weight of active enzyme of the composition Such liquid fabric cleaning compositions typically additionally comprise an anionic surfactant, a fatty acid, a water-solubie detergency builder and water

The liquid fabric cleaning composition embodiment of the present invention is illustrated by the following examples Examples 48-52 Liquid Fabric Cleaning Compositions

In Examples 48-50 the Thermitase variants recited in Tables 2-36, among others, are substituted for Asn166Asp + Thr167Asn + Asn170Ser + Tyr171Thr, with substantially similar results.

In Examples 51-52, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Asπ166Asp + Thr167Asn + Asn170Ser + Tyr171Thr and Ser191 Glu + Ser192Asp + Phe193Asp + Thr195Pro, with substantially similar results.

Examples 53-57 Liquid Fabric Cleaning Compositions

In Examples 53-55 the Thermitase variants recited in Tables 2-36, among others, are substituted for Pro205Gln + Gly206Ser + Trp208Val + Tyr210Leu + Thr212Asn + Tyr218Met + Ser220Glu, with substantially similar results

In Examples 56-57 any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Pro205Glπ + Gly206Ser + Trp208Val + Tyr210Leu + Thr212Asn + Tyr218Met + Ser220Glu and Asn 68Asp + Val138Asp + Ala164Gly + Ala168Glu + Thr217Gly, with substantially similar results

Examples 58-59 Liquid Fabric Cleaning Composition

In each of Examples 58 and 59 herein, the Thermitase variants recited in Tables 2-36, among others, are substituted for Leu134Cys + Thr137Glu + Val138Giy, with substantially similar results Examples 60-62 Liquid Fabric Cleaning Composition

Bar fabric cleaning compositions of the present invention suitable for hand-washing soiled fabrics contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0 001 % to about 10%, more preferably from about 0 01 % to about 1 % by weight of the composition

The bar fabric cleaning composition embodiment of the present invention is illustrated by the following examples Examples 63-66 Bar Fabric Cleaning Compositions

^*Can be selected from convenient materials such as CaCO₃, talc, clay, silicates, and the like

In Examples 63-64 the Thermitase variants recited in Tables 2-36, among others, are substituted for Trp112Cys + Thr217Gly, with substantially similar results.

In Examples 65-66, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Trp112Cys + Thr217Gly and Val103Ala + Thr212Ser, with substantially similar results Examples 67-70 Bar Fabric Cleaning Compositions

In Example 67, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser109Glu, with substantially similar results

In Example 68, the Thermitase variants recited in Tables 2-36, among others, are substituted for Pro169Glu, with substantially similar results

In Examples 69-70, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser109Glu and Pro169Glu, with substantially similar results B Additional Cleaning Compositions

In addition to the hard surface cleaning, dishwashing and fabric cleaning compositions discussed above, one or more enzyme variants of the present invention may be incorporated into a variety of other cleaning compositions where hydrolysis of an insoluble substrate is desired Such additional cleaning compositions include but are not limited to, oral cleaning compositions, denture cleaning compositions, and contact lens cleaning compositions

1 Oral cleaning compositions

In another embodiment of the present invention, a pharmaceutically- acceptable amount of one or more enzyme variants of the present invention are included in compositions useful for removing proteinaceous stains from teeth or dentures As used herein, "oral cleaning compositions" refers to dentifrices, toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxis pastes, dental treatment solutions, and the like Preferably, the oral cleaning compositions comprise from about 0 0001 % to about 20% of one or more enzyme variants of the present invention, more preferably from about 0 001 % to about 10%, more preferably still from about 0 01 % to about 5%, by weight of the composition, and a pharmaceutically-acceptable carrier As used herein, "pharmaceutically-acceptable" means that drugs, medicaments or inert ingredients which the term describes are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio

Typically, the pharmaceutically-acceptable oral cleaning carrier components of the oral cleaning components of the oral cleaning compositions will generally comprise from about 50% to about 99 99%, preferably from about 65% to about 99 99%, more preferably from about 65% to about 99%, by weight of the composition

The pharmaceutically-acceptable carrier components and optional components which may be included in the oral cleaning compositions of the present invention are well known to those skilled in the art A wide variety of composition types, carrier components and optional components useful in the oral cleaning compositions are disclosed in U S Patent 5,096,700, Seibel, issued March 17, 1992, U S Patent 5,028,414, Sampathkumar, issued July 2, 1991 , and U S Patent 5,028,415, Benedict, Bush and Sunberg, issued July 2, 1991 , all of which are incorporated herein by reference

The oral cleaning composition embodiment of the present invention is illustrated by the following examples Examples 71 -74 Dentifrice Composition

Example No.

Component 71 72 73 74

Tyr218Ala 2.000 3.500 1.500 2.000

Sorbitol (70% aqueous solution) 35.000 35.000 35.000 35.000

PEG-6* 1 .000 1 .000 1.000 1.000

Silica dental abrasive^** 20.000 20.000 20.000 20.000

Sodium fluoride 0.243 0.243 0.243 0.243

Titanium dioxide 0.500 0.500 0.500 0.500

Sodium saccharin 0.286 0.286 0.286 0.286

Sodium alkyl sulfate (27.9% 4.000 4.000 4.000 4.000 aqueous solution) Flavor 1.040 1.040 1.040 1.040

Carboxyvinyl Polymer^*** 0.300 0.300 0.300 0.300 Carrageenan^* 0.800 0.800 0.800 0.800 Water balance to 100%

*PEG-6 = Polyethylene glycol having a molecular weight of 600. **Precipitated silica identified as Zeodent 119 offered by J.M. Huber. ***Carbopol offered by B.F. Goodrich Chemical Company. ****lota Carrageenan offered by Hercules Chemical Company.

In Examples 71 -74 the Thermitase variants recited in Tables 2-36, among others, are substituted for Tyr218Ala, with substantially similar results.

Examples 75-78 Mouthwash Composition

In Examples 75-78, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ala164Hιs + Ser220Glu, with substantially similar results

Examples 79-82 Lozenge Composition

Example No

Component 79 80 81 82

Leu221Val + Thr224Gln

Sorbitol

Mannitol

Starch

Sweetener

Flavor

Color

Corn Syrup

In Examples 79-82, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser Leu221Val + Thr224Gln, with substantially similar results Examples 83-86 Chewing Gum Composition

Example No

Component 83 84 85 86

Ser191Asp + Phe193lle + Thr195Gly 0 03 0 02 0 10 0 05

Sorbitol crystals

Paloja-T gum base^*

Sorbitol (70% aqueous solution)

Mannitol

Glycerine

Flavor

*Supplιed by L A Dreyfus Company

In Examples 83-86, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser191Asp + Phe193lle + Thr195Gly, with substantially similar results 2 Denture cleaning compositions

In another embodiment of the present invention, denture cleaning compositions for cleaning dentures outside of the oral cavity comprise one or more enzyme variants of the present invention Such denture cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0 0001 % to about 50% of one or more of the enzyme variants, more preferably from about 0 001 % to about 35%, more preferably still from about 0 01% to about 20%, by weight of the composition, and a denture cleansing carrier Various denture cleansing composition formats such as effervescent tablets and the like are well known in the art (see for example U S Patent 5,055,305, Young, incorporated herein by reference), and are generally appropriate for incorporation of one or more of the enzyme variants for removing prote aceous stains from dentures

The denture cleaning composition embodiment of the present invention is illustrated by the following examples Examples 87-90 Two-layer Effervescent Denture Cleansing Tablet

Tetraacetylethylene diamine

In Examples 87-90, the Thermitase variants recited in Tables 2-36, among others, are substituted for Gly135Gln + Gly139Asn + Asn140Gln + Ser141Asp, with substantially similar results. 3. Contact Lens Cleaning Compositions

In another embodiment of the present invention, contact lens cleaning compositions comprise one or more enzyme variants of the present invention. Such contact lens cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0.01 % to about 50% of one or more of the enzyme variants, more preferably from about 0 01 % to about 20%, more preferably still from about 1 % to about 5%, by weight of the composition and a contact lens cleaning carrier Various contact lens cleaning composition formats such as tablets, liquids and the like are well known in the art (see for example U S Patent 4,863,627, Davies, Meaken and Rees, issued September 5, 1989, U S Patent Re 32,672, Huth, Lam and Kirai, reissued May 24 1988, U S Patent 4 609 493 Schafer, issued September 2 1986 U S Patent, 4,690,793, Ogunbiyi and Smith, issued September 1 , 1987, U S Patent 4,614,549, Ogunbiyi, Riedhammer and Smith, issued September 30, 1986, and U S Patent 4,285,738, Ogata, issued August 25, 1981 , each of which are incorporated herein by reference), and are generally appropriate for incorporation of one or more enzyme variants of the present invention for removing proteinaceous stains from contact lens

The contact lens cleaning composition embodiment of the present invention is illustrated by the following examples

Examples 91-94 Enzymatic Contact Lens Cleaning Solution

In Examples 91-94, the Thermitase variants recited in Tables 2-36, among others, are substituted for Leu221 Gln, with substantially similar results

While particular embodiments of the subject invention have been described, it will be obvious to those skilled in the art that various changes and modifications of the subject invention can be made without departing from the spirit and scope of the invention It is intended to cover, in the appended claims all such modifications that are within the scope of the invention

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: BRODE, PHILIP F. BARNETT, BOBBY L. RUBINGH, DONN N.

(ii) TITLE OF INVENTION: THERMITASE VARIANTS HAVING DECREASED ADSORPTION AND INCREASED HYDROLYSIS

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: THE PROCTER & GAMBLE COMPANY

(B) STREET: 11810 East Miami River Road

(C) CITY: Ross

(D) STATE: OH

(E) COUNTRY: USA

(F) ZIP: 45061

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: CORSTANJE, BRAHM J.

(B) REGISTRATION NUMBER: 34,804

(C) REFERENCE/DOCKET NUMBER: 5607

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (513) 627-2858

(B) TELEFAX: (513) 627-0260

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 279 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Tyr Thr Pro Asn Asp Pro Tyr Phe Ser Ser Arg Gin Tyr Gly Pro

Gin

1 5 10 15

Lys lie Gin Ala Pro Gin Ala Trp Asp lie Ala Glu Gly Ser Gly

Ala

20 25 30

Lys lie Ala lie Val Asp Thr Gly Val Gin Ser Asn His Pro Asp

Leu

35 40 45

Ala Gly Lys Val Val Gly Gly Trp Asp Phe Val Asp Asn Asp Ser

Thr 50 55 60

Pro Gin Asn Gly Asn Gly His Gly Thr His Cys Ala Gly He Ala

Ala

65 70 75 80

Ala Val Thr Asn Asn Ser Thr Gly He Ala Gly Thr Ala Pro Lys

Ala

85 90 95

Ser He Leu Ala Val Arg Val Leu Asp Asn Ser Gly Ser Gly Thr Trp

100 105 110

Thr Ala Val Ala Asn Gly He Thr Tyr Ala Ala Asp Gin Gly Ala Lys

115 120 125

Val He Ser Leu Ser Leu Gly Gly Thr Val Gly Asn Ser Gly Leu Gin

130 135 140

Gin Ala Val Asn Tyr Ala Trp Asn Lys Gly Ser Val Val Val Ala Ala

145 150 155

160

Ala Gly Asn Ala Gly Asn Thr Al Pro Asn Tyr Pro Ala Tyr Tyr Ser

165 170 175

Asn Ala He Ala Val Ala Ser Thr Asp Gin Asn Asp Asn Lys Ser Ser

180 185 190

Phe Ser Thr Tyr Gly Ser Val Val Asp Val Ala Ala Pro Gly Ser Trp

195 200 205

He Tyr Ser Thr Tyr Pro Thr Ser Thr Tyr Ala Ser Leu Ser Gly Thr

210 215 220

Ser Met Ala Thr Pro His Val Ala Gly Val Ala Gly Leu Leu Ala Ser

225 230 235

240

Gin Gly Arg Ser Ala Ser Asn He Arg Ala Ala He Glu Asn Thr Ala

245 250 255

Asp Lys He Ser Gly Thr Gly Thr Tyr Trp Ala Lys Gly Arg Val Asn

260 265 270

Ala Tyr Lys Ala Val Gin Tyr

275

Claims

What is Claimed is

1 A Thermitase variant having a modified ammo acid sequence of

Thermitase wild-type ammo acid sequence, the wild-type ammo acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region, wherein the modified ammo acid sequence comprises a substitution at one or more positions in one or more of the loop regions, wherein

A when a substitution occurs in the first loop region, the substitution occurs at one or more of positions 66, 69, 70, 72 or 73, wherein a when a substitution occurs at position 66, the substituting ammo acid is Asn, b when a substitution occurs at position 69, the substituting ammo acid is Gin, c when a substitution occurs at position 70, the substituting am o acid is Asn, Asp, Gin, Glu, Pro or Ser, d when a substitution occurs at position 72, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser, and e when a substitution occurs at position 73, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser, B when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 103, 104, 105, 106, 108, 1 10, 1 12, 1 13 or 1 14, wherein a when a substitution occurs at position 103, the substituting am o acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr, b when a substitution occurs at position 104, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Gly, His, lie, Met, Pro or Ser, c when a substitution occurs at position 105, the substituting ammo acid is Glu, d when a substitution occurs at position 106, the substituting ammo acid is Gin, e when a substitution occurs at position 108, the substituting ammo acid is Asn, Gin, Pro or Ser, f when a substitution occurs at position 110, the substituting am o acid is Asn, Asp, Gin, Glu, Pro or

Ser, g when a substitution occurs at position 112, the substituting am o acid is Asn, Asp, Cys, Gin, Glu, His, lie, Met, Phe, Pro, Thr or Tyr, h when a substitution occurs at position 113, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or

Ser, and i when a substitution occurs at position 114, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, His,

Pro, Ser or Thr, C when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 134, 135, 136, 137, 138, 139 or 141 , wherein a when a substitution occurs at position 134, the substituting ammo acid is Ala, Asn, Cys, Gin, Gly, His,

Met, Pro, Ser, Thr or Val, b when a substitution occurs at position 135, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or

Ser, c when a substitution occurs at position 136, the substituting ammo acid is Pro, d when a substitution occurs at position 137, the substituting ammo acid is Asn, Asp, Gin, Glu or Ser, e when a substitution occurs at position 138, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu,

Gly, His, Met, Pro, Ser or Thr, f when a substitution occurs at position 139, the substituting am o acid is Asn, Gin, Pro or Ser, and g when a substitution occurs at position 141 , the substituting am o acid is Asp or Glu, D when a substitution occurs in the fourth ioop region, the substitution occurs at one or more of positions 167, 169, or 171 , wherein a when a substitution occurs at position 167, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or

Ser, b when a substitution occurs at position 169, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly or

Ser, and c when a substitution occurs at position 171 , the substituting ammo acid is His, lie, Leu, Met or Pro, E when a substitution occurs in the fifth loop region, the substitution occurs at one or more of position 193, wherein a when a substitution occurs at position 193, the substituting ammo acid is Asn, Cys, Gin, His, He, Met,

Thr or Tyr, F when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 204, 205 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 220, 221 , 223 or 224, wherein a when a substitution occurs at position 204, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, His,

Pro, Ser or Thr, b when a substitution occurs at position 205, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly or

Ser, c when a substitution occurs at position 206, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or

Ser, d when a substitution occurs at position 207, the substituting am o acid is Asp or Glu, e when a substitution occurs at position 208, the substituting ammo acid is Asp or Glu, f when a substitution occurs at position 209, the substituting am o acid is Ala, Asn, Asp, Cys, Gin, Glu,

Gly, His, Leu, Met, Pro, Ser, Thr or Val, g when a substitution occurs at position 210, the substituting ammo acid is Asp, His, He, Leu, Met or Pro, h when a substitution occurs at position 211 , the substituting ammo acid is Asp or Glu, i when a substitution occurs at position 212, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or

Ser, j when a substitution occurs at position 213, the substituting am o acid is Asp, Glu, His, He, Leu, Met,

Phe, Pro or Val, k when a substitution occurs at position 214, the substituting am o acid is Asn, Gin, Gly, or Ser, I when a substitution occurs at position 215, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or

Ser m when a substitution occurs at position 216, the substituting am o acid is Asp or Glu, n when a substitution occurs at position 217, the substituting ammo acid is Asp, Glu or Pro, o when a substitution occurs at position 220, the substituting ammo acid is Asp or Glu, p when a substitution occurs at position 221 , the substituting am o acid is Ala, Asn, Asp, Cys, Gin, Glu,

Gly, His, lie, Met, Pro, Ser, Thr or Val, q when a substitution occurs at position 222, the substituting am o acid is Asp or Glu, r when a substitution occurs at position 223, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or

Ser, and s when a substitution occurs at position 224, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or

Ser, whereby the Thermitase variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Thermitase

2 The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the first loop region

3 The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the second loop region 4 The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the third loop region

5 The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the fourth loop region

6 The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the fifth loop region

7 The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the sixth loop region

8 A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, characterized in that the cleaning composition comprises the Thermitase variant of any of Claims 1 -7 and a cleaning composition carrier, preferably the cleaning composition is a hard surface cleaning composition or the cleaning composition is a fabric cleaning composition, preferably the composition comprises at least about 5% surfactant and at least about 5% builder, by weight of the composition, preferably the composition further comprises cleaning composition materials selected from the group consisting of solvents, buffers, enzymes, soil release agents, clay soil removal agents dispersing agents, brighteners, suds suppressors, fabric softeners, suds boosters, enzyme stabilizers, bleaching agents, dyes, perfumes, and mixtures thereof

9 A DNA sequence encoding the Thermitase variant of any of Claims

1-7