WO1993015110A1 - Sequences d'acides amines s'appariant de maniere specifique - Google Patents

Sequences d'acides amines s'appariant de maniere specifique Download PDF

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Publication number
WO1993015110A1
WO1993015110A1 PCT/US1993/000884 US9300884W WO9315110A1 WO 1993015110 A1 WO1993015110 A1 WO 1993015110A1 US 9300884 W US9300884 W US 9300884W WO 9315110 A1 WO9315110 A1 WO 9315110A1
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Prior art keywords
amino acid
peptide
reεidueε
residues
residueε
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PCT/US1993/000884
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English (en)
Inventor
Erin K. O'shea
Peter S. Kim
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Whitehead Institute For Biomedical Research
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Publication of WO1993015110A1 publication Critical patent/WO1993015110A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)

Definitions

  • the present invention relates to pairs of synthetic peptides designed in such a manner that they pair specifi ⁇ cally with one another to form a heterodimer and then, once paired, preferentially fold as a helical heterodimer.
  • the present invention further relates to a method of making pairs of synthetic peptides which bind prefer ⁇ entially to one another and to methods of producing such peptide pairs.
  • the peptide members of the pair may be of any length, provided they are sufficiently long that they are stable in the heterodimeric form and are able to fold into a helical configuration.
  • the two members of a pair will " typically be of the same length, although this is not required.
  • the individual peptides will be at least 6 to 8 amino acid residues in length, generally at least 12-14 amino acid residues in length and will prefer ⁇ ably be at least 16-20 amino acid residues in length. In one embodiment, the individual peptides will be at least 20-23 amino acids in length. In another embodiment, the individual peptides will be 24-30 amino acids in length, particularly to 30 amino acids in length. There is no upper limit on individual peptide length. More than one peptide "repeat" or unit may be combined in a peptide pair of the present invention, if desired.
  • the second member of the peptide pair also includes multiple peptide repeats or units, whose amino acid sequences are designed to pair specifically and avidly with the first peptide repeats and preferentially form a coiled-coil helical heterodimer.
  • it relates to pairs of synthetic peptides (whose members are designated ACID-pl and BASE-pl herein and A-l and B-l in U.S. Application, Serial No.
  • homodimers of the synthetic peptides are very unstable, relative to heterodimers of the synthetic peptides and, in an equil ⁇ ibrium mixture of the two peptides, the heterodimer is favored over the homodimers by at least 1-million-fold.
  • two peptides designated A-l and B-l in U.S. Application,
  • Serial No. 07/829,140 designed to bind one another spec ⁇ ifically and avidly and to preferentially form a helical heterodimer have been synthesized and characterized, using known methods. As described herein, under physiological conditions, individual peptides have been shown, using circular dichrois (CD) spectroscopy, to be predominantly unfolded in isolation and when combined, to associate preferentially to produce stable, parallel, coiled-coil (helical) heterodimers. Further, the degree of preference for the heterodimers has been estimated by studying the stability of the heterodimers and homodimers, using CD methods; results showed that the heterodimer has much greater stability than either of the homodimers.
  • CD circular dichrois
  • the observed difference in stability between the homodimers and the heterodimer suggests that the heterodimer is preferred over the homodimers by at least 1-million-fold.
  • the oligomerization state, helical content and helix orientation can be assessed, using, respective ⁇ ly, sedimentation equilibrium studies, CD and disulfide bonding of the peptides in the desired parallel orient ⁇ ation, followed by measurement of the concentration depen ⁇ dence of stability.
  • two peptides each containing two and 14 amino acid re- peats designated, respectively, ACID-pl and BASE-pl, (designated A-l and B-l in a related U.S. Application, Serial No. 07/829,140) were designed to bind to one another specifically and avidly and to preferentially form a helical heterodimer.
  • the two peptides have been syn- thesized and characterized using known methods.
  • the individual peptides (ACID-pl and BASE-pl) were shown to be pre ⁇ dominantly unfolded and an equimolar mixture of ACID-pl and BASE-pl has been shown to preferentially form hetero ⁇ dimers which are stable, parallel in orientation and highly helical.
  • the heterodimer has been shown to have greater stability than either of the homodimers.
  • the amino acid composition and order of the constituent pep- tides were designed to introduce destabilizing electro- static interactions in the homodimers that would be re ⁇ lieved in the heterodimers.
  • the two peptides differ only at two positions (designated e and g, as defined below) by a single amino acid.
  • the ACID-pl peptide contains, at these two positions, an acidic amino acid, such as glu- tamic acid; the BASE-pl peptide contains, at these two positions, a basic amino acid, such as lysine.
  • Measure ⁇ ments of the specificity of dimer formation demonstrate that these peptides have at least 10 5 -fold preference for the heterodimeric state.
  • Studies of the pH and ionic strength dependence of stability confirm that electro ⁇ static destabilization of the homodimers provides the primary driving force for the specificity of heterodimer formation.
  • the synthetic peptide pairs of the present invention can be used as affinity reagents to isolate associated molecules.
  • a synthetic peptide pair such as ACID-pl and BASE-pl, can be used in place of biotin-avidin (e.g., in a biotin/streptavidin affinity method), epitope tagging or immunoaffinity purification methodology.
  • the syn ⁇ thetic peptides of the present invention are suitable for use in vivo.
  • one member of the synthetic peptide pair e.g., ACID-pl
  • chimeric peptide 1 which includes the synthetic peptide (one member of the peptide pair) and an additional com ⁇ ponent which is not a synthetic peptide of the present invention.
  • the second component can be a peptide, poly- peptide, glyco- or other protein, a detectable label or a small organic molecule, which is to be joined or brought into contact with another molecule.
  • the other member of the synthetic peptide pair can be, for example, included in a second chimeric or hybrid product (chimeric peptide 2) which includes the synthetic peptide (e.g., BASE-pl) and the molecule with which the additional component of the first chimeric product (chimeric peptide 1) is to bind or otherwise interact with.
  • chimeric or fusion peptides are also the subject of this invention. They can be produced using known techniques, such as recombinant production methods or chemical (synthetic) methods. They can be produced as one product (e.g., a chimeric peptide which includes both components as produced) or the com- ponents can be produced individually and then joined, using known methods.
  • the synthetic peptide pairs can be used, for example, for in vivo applications in which two molecules or components necessary for a given event must be brought together for the event to occur.
  • the synthetic peptide pairs of the present invention make it possible to bring the components together with great specificity and affinity.
  • the synthetic peptide pairs can be designed to prevent binding or interaction of molecules (e.g., DNA and its DNA binding protein) necessary for an event to occur.
  • the synthetic peptide pairs can also be used for biodegradable procedures involving grafting and artificial sutures.
  • Figure 1 is a coiled-coil helical wheel represen- tation of amino acid residues present in peptide A and peptide B at the locations indicated.
  • Figure 2 is a coiled-coil wheel representation of the sequence of the ACID-pl/ACID-pl peptide homodimer (Panel A) , the BASE-pl/BASE-pl peptide homodimer (Panel B) and the ACID-pl/BASE-pl peptide heterodimer of the present invention (Panel C) .
  • the sequence of the peptide ACID-pl and peptide BASE-pl is arrayed on a coiled-coil diagram in which the helices of the dimer are viewed from the N-terminus (with the helix axis projecting into the page) .
  • the sequence of the coiled-coil peptide is divided into positions of the heptad repeat, labeled a- g; amino acid residues at positions a and d make up the 4- 3 hydrophobic repeat characteristic of coiled coils. Amino acids are represented by their respective one-letter cod .
  • Figure 3 presents circular dichroism spectra of the ACID-pl peptide, the BASE-pl peptide and the ACID-pl/BASE- pl heterodimer at 37°C in phosphate buffered saline (PBS) , pH 7.0 (physiological conditions).
  • PBS phosphate buffered saline
  • Figure 4 presents a circular dichroism melting curve of the ACID-pl/BASE-pl heterodimer at a wavelength of 222nm.
  • the present invention is based on Applicant's dis ⁇ covery of characteristics of pairs of peptides necessary and sufficient for such peptides to bind or pair specific ⁇ ally and avidly to one another, to form heterodimers, and for the resulting heterodimers to preferentially fold as a helical heterodimer.
  • peptide pair members which, when mixed, associate preferentially to form a stable, parallel coiled-coil heterodimer, rather than their respective homodimeric forms.
  • the peptides of the present invention may be of any length, provided that they are sufficiently long to be stable when paired with the other member of the peptide pair (i.e., in the hetero ⁇ dimeric form) and to be able to allow the heterodimer to fold into a helical configuration.
  • the pep- tides will be at least 6 to 8 amino acid residues, gener ⁇ ally at least 12 to 14 amino acid residues in length and preferably, at least 16 to 20 amino acid residues in length or between 20 or 30 amino acid residues long.
  • each member of the peptide pair has two 14 amino acid residue "repeats" (two 14 amino acid units) resulting in each pair member being 28 amino acids long. In another embodiment, each pair member is 30 amino acids long. In another embodiment, each pair member is 100 amino acids long. There is no upper limit on the length of peptide members and the appropriate length will be determined by such considerations as the context in which a peptide pair is to be used (e.g., n vitro or in vivo, isolated method, therapeutic or diagnostic techniques) . More than one peptide repeats or units of the present invention can be combined (i.e., present in a single multi-unit peptide) if desired.
  • more than one repeat peptide designed to pair specifically and avidly to a second peptide can be combined in a first multi- unit peptide and more than one repeat of the second pep ⁇ tide, (e.g., designated peptide BASE-pl herein and B-l in U.S. Application Serial No. 07/829,140) also described herein, can be combined in a second multi-unit peptide and the wo multi-unit peptides used as the two members of the peptide pair.
  • the individual peptide units in the multi- unit peptide can be the same or different.
  • the individual peptide units differ within a peptide (e.g., there are two or more peptide units which are not identical in sequence) but are the same between peptides (e.g., the two members of the pep ⁇ tide pair each contain the same peptide unit(s) as the other member) .
  • Amino acids in the peptides can be natu- rally-occurring amino acids, non-naturally-occurring amino acids or modified amino acids.
  • the members of a peptide pair stick avidly to the other member of the pair, but do not bind efficiently to a like member of the pair.
  • Figures 1 and 2 are coiled-coil helical wheel repre ⁇ sentations of 30 amino acid residues of the ACID-pl/ACID- pl peptide homodimer ( Figure 2, Panel A) , the BASE-PI/ BASE-PI peptide homodimer ( Figure 2, Panel B) and the ACID-pl/BASE-pl peptide heterodimer ( Figure 2, Panel C) .
  • the sequence of the coiled-coil peptide represented is divided into positions of the heptad repeat, which are labeled a through g. Selection of the appropriate type of amino acid residue for each of the positions relied on studies of naturally-occurring leucine zipper peptides, particularly the Fos/Jun leucine zipper heterodimer and the GCN4 leucine zipper homodimer, as described below.
  • the peptide pairs described herein were designed based on principles learned from Applicant's study of coiled-coil amino acid sequences, including the hetero- dimeric Fos/Jun leucine zipper.
  • the peptide pairs are designed in such a manner that, in isolation, the indi ⁇ vidual peptides are unfolded and do not join or stick to a like peptide efficiently (i.e., two of the same peptide pair members do not stick efficiently together to form homodimers) .
  • the pep ⁇ tide pair members join preferentially to form a stable heterodimeric coiled-coil.
  • the primary driving force for the specificity of binding to form a heterodimer is elec ⁇ trostatic destabilization of the homodimers.
  • the designed peptides fold as parallel, helical dimers which have a great (at least about 10 5 -fold) preference for the heterodimeric state.
  • charged amino acid residues are present at positions e and g (and e' and g » ; Figure 1).
  • a negatively charged amino acid such as glutamate (glutamic acid)
  • a positively charged amino acid such as lysine
  • a negatively charged amino acid residue is present at e (amino acid residues 4, 11, 18, 25) and g (amino acid residues 6, 13, 20, 27) in peptide ACID-pl and a positively charged amino acid is present at e » (amino acid residues 4, 11, 21, 25) and g* (amino acid residues 6, 13, 20, 27) in peptide BASE-pl.
  • a hydrophobic amino acid, such as leucine is present at positions a and d (peptide ACID-pl) and a 1 and d' (peptide BASE-pl) .
  • Leucine is a preferred choice because it is the most common amino acid at these positions in naturally- occurring coiled coils.
  • an asparagine is present at the second a posi ⁇ tion (in peptide ACID-pl, position a and in peptide BASE- pl, position a 1 , both of which represent amino acid 14) in order to favor the parallel orientation and discourage higher order oligomerization.
  • Asparagine is present at the corresponding position in the GCN4 leucine zipper, which is a peptide which folds as a two-stranded parallel coiled coil.
  • amino acid residues present at positions b, c and f, as represented in Figure 1 can be very varied; almost any combination of amino acid residues can be used, provided that there is an appropriate distribution of hydrophilic amino acid residues at these positions. Selection of amino acid residues appropriate for inclusion at positions b, c and f can be made with reference to Applicants' work (e.g., Example 1) and work by Conway and Parry (see above) .
  • small, uncharged amino acid residues are present at positions b and c and b' and c'.
  • Such small, uncharged amino acid residues as alanine and glutamine are used at these locations in order to prevent residues at b and c and b 1 and c* from interacting with residues at positions e and g and e' and g' and, as a result, co - peting with the desired interhelical interactions.
  • polar residues included in the peptide pair members increase solubility of the peptide.
  • charged residues such as glutamate and ly- sine
  • position f and f, see Figure 1
  • a single tryptophan at position f and f• has been included in peptides ACID-pl and BASE-PI as a means of facilitating concentration determination by absorbance.
  • they will generally be a charged residue, such as glutamate or lysine.
  • the two members (peptide ACID-pl and peptide BASE-pl) of the synthetic peptide pairs of the present invention have been a general formula which can be des ⁇ cribed with reference to the coiled-coil helical wheel representation shown in Figure 1.
  • the following amino acid residues are present:
  • hydrophobic amino acid residue such as leucine
  • hydrophilic amino acids such as a negatively charged amino acid residue (e.g., alanine, glutamine) at b' and c* and such as a positively charged amino acid residue (e.g., glutamate, lysine) at f' .
  • proline will not be used in these peptides.
  • amino acid residues present at each of these positions in one embodiment of the two pair members are shown in Figure 2 (by their one-letter code) and in Table 1 (peptide ACID-pl, peptide BASE-pl, respectively, by their three-letter codes) .
  • the amino acid residues can additionally include Cys-Gly- Gly, which is generally added at the N-terminal of the peptide and not part of the 30 amino acid residue peptide of the present invention but, rather, is included for assay purpose ⁇ only.
  • Cy ⁇ -Gly-Gly can be added to the peptide (before amino acid re ⁇ idue 1) to assi ⁇ t in the a ⁇ e ⁇ sment of helix orientation (parallel vs. antiparallel) of the heterodimer.
  • a disulfide-bonded peptide (disulfide-bonded in parallel orientation) is assessed by measuring its stability as a function of peptide concentration; alternatively, molecu ⁇ lar weight can be measured as a function of peptide con ⁇ centration by sedimentation. If the helices are parallel, the disulfide-bonded peptide is expected to have stability independent of peptide concentration and molecular weight equal to the molecular weight of the dimer independent of peptide concentration.
  • the members of the peptide pairs of the present invention can be produced using known methods, such a ⁇ chemical synthesi ⁇ or recombinant/genetic engineering technology. For example, they can be synthesized as described in Example 2 or in much the same manner as described in Example 1 for the synthesis of the Fos and Jun leucine zipper peptides. Alternatively, the peptides can be produced in an appropriate host cell by expres ⁇ ing DNA or RNA encoding the peptide sequence. As used herein, the term synthetic refers to peptides of the present invention made by any method (e.g., chemically or by recombinant or genetic engineering methods) . Peptide pair ⁇ of the pre ⁇ ent invention have many uses, in both in vitro and in vivo contexts.
  • peptide pairs can be used as affinity reagents, in much the same way as or as a replacement for other binding pairs.
  • they can be used in place of biotin-streptavidin, epitope tagging methods or immunoseparation methods.
  • a member of a peptide pair can be attached or linked to another molecule (e.g., another peptide, polypeptide, glyco- or other protein, or a detectable label or small organic molecule) or to a solid support (e.g., a column, particle, filter, plastic plate) by known methods, such as a component of a fusion protein or through a linker (e.g., the Cys/Gly/Gly referred to above) for attachment to a solid surface.
  • another molecule e.g., another peptide, polypeptide, glyco- or other protein, or a detectable label or small organic molecule
  • a solid support e.g., a column, particle, filter, plastic plate
  • a molecule to which the second member of the peptide pair is attached can be separated or isolated by contacting a mixture containing the molecule-peptide pair with the solid surface bearing the second member of the peptide pair, under conditions appropriate for sticking or pairing of the peptide pair members.
  • the fraction of the mixture which is not the molecule to be separated or isolated will not become affixed to the solid support and can be removed simply by separating the solid support from the remainder of the mixture.
  • the bound molecule (bound as a result of pairing of the peptide pair members) can be released from the solid support by, for example, changing the pH and/or temperature of the bound fraction.
  • Such a method of separating or isolating a molecule in this manner can be used, for example, for purification of a molecule to be used for other purpo ⁇ e ⁇ (e.g. , where presence of a par ⁇ ticular substance is indicative of the pre ⁇ ence or absence of a disea ⁇ e or condition) .
  • Peptide pairs of the present method can al ⁇ o be u ⁇ e for in vivo purpo ⁇ e ⁇ , such a ⁇ to block, induce or enhance an event in cell ⁇ (e.g., to interfere with binding of two components in a cell where binding is neces ⁇ ary, thus inducing or enhancing the event) .
  • peptides of the present invention can be produced in cells in which they are to act by, for example, expression from a vector, such a ⁇ a retroviral vector( ⁇ ) containing DNA or RNA encoding a chimeric peptide or peptide ⁇ .
  • the chimeric peptide in ⁇ cludes the amino acid residues of the peptide of the present invention and, if desired, a peptide or a poly- peptide which is not a synthetic peptide of the present invention, such a ⁇ a peptide which is to act in the cell.
  • peptides of the present invention can be used for radioimaging or to treat diseases, such as malig- nancies.
  • one member of a peptide pair i ⁇ expressed in the malignant cell e.g., from an appropriate vector
  • the second member of the peptide pair can be labeled, thereby capable of detection or can be joined with an agent which is capable of detec- tion (e.g., a radioactive molecule or substance, such as ricin, toxic to cells) which binds specifically to the cell expres ⁇ ing the fir ⁇ t member of the peptide pair.
  • an agent which is capable of detec- tion e.g., a radioactive molecule or substance, such as ricin, toxic to cells
  • Such an approach can also be used, for example, in treat ⁇ ing hyperthyroidism.
  • One member of the peptide pair can be expre ⁇ ed (e.g., from a retroviral or other vector) in the thyroid and the second member of the peptide pair joined to radioactive iodine can be administered to an individual in need of treatment. Pairing of the two peptide pair members results in delivery of the radio- active iodine to the thyroid; continuous delivery occur ⁇ until the peptide pair is degraded by the body or other ⁇ wise ' separated.
  • peptide pairs can be joined to other peptides or proteins (e.g., peptides or proteins to be delivered) by chemical means or included with/incorporated into another peptide or protein which is made by recombi- nant DNA methods.
  • a peptide pair member may be joined or present at an end of the peptide or protein or at any internal site at which the protein can tolerate insertion of the peptide pair member (i.e., any site at which the peptide pair member can be present and not interfere with the desired function of the other peptide or protein) .
  • Peptides were ⁇ ynthesized using t-BOC chemistry on an Applied Biosystems model 430A peptide synthe ⁇ izer with ⁇ tandard reaction cycle ⁇ modified to include acetic an- hydride capping (for a review ⁇ ee Kent, S.B.H., Annu. Rev. Bioche . 57:956-989 (1988).
  • Peptide Jun N corresponds to residue ⁇ 286-317 of the c-Jun protein (Bohmann, D. et al.. Science 238:1386-1392 (1987); Maki, Y. ⁇ £ Al s ., proc. Natl. Acad. Sci.
  • peptide Fo ⁇ N corresponds to residue ⁇ 162-193 of the c-Fo ⁇ protein (Van Beveren, C. et aJ , Cell 32:1241-1255 (1983); Van Straaten, F. et al.. Proc. Natl. Acad. Sci. USA 80:3183- 3187 (1983)).
  • Ser-295 of c-Jun and Ser-177 of c-Fo ⁇ have been replaced with tyro ⁇ ine to facilitate concentration determination by UV ab ⁇ orbance measurements.
  • Peptide GCN4 N con ⁇ i ⁇ ts of residues 250-281 of the GCN4 protein (Hinne- bu ⁇ ch, A.G., Proc. Natl.
  • All peptides have the sequence Cys-Gly-Gly appended to the N-terminus, are acetylated at the N-termi- nus, and are amidated at the C-terminus.
  • Peptide ⁇ were cleaved by either low/high HF cleavage (Immunodynamic ⁇ , Inc., San Diego, CA) or by trifluoromethanesulfonic acid cleavage (Kent, S.B.H., Annu. Rev. Biochem. 57:956-989 (1988) and were desalted on a Sephadex G-10 column (Pharm- acia) in 5% acetic acid.
  • each peptide was confirmed by fast atom bom ⁇ bardment mass spectrometry (M-Scan, Inc., West Chester, PA or Mas ⁇ Search, Inc., Modesto, CA) and was found to be within 1 dalton of the expected mass.
  • Circular dichroism (CD) studies were performed using a 1 cm or 1 mm cuvette (Helma or Uvonic) on an Aviv CD spectrophotometer (model 60DS or model 62DS) equipped with a thermoelectric controller.
  • the buffer used for all CD experiments except pH titrations was 50 mM NaCl, 20 mM NaP0 4 (pH 7.0). All peptide concentrations were deter ⁇ mined by tyrosine ab ⁇ orbance (Edelhoch, H.
  • the T m was determined by curve fitting the thermal denaturation curve to the following equation using a nonlinear least squares-fitting program (Kaleidagraph,
  • T temperature in K
  • ⁇ is the CD signal at 222 nm
  • ⁇ F (O K) is the value for the CD signal of the folded peptide extrapolated linearly to 0 K
  • m F is the slope of the temperature dependence of the CD signal for the folded peptide
  • m y is the slope of the temperature dependence of the CD signal for the unfolded peptide
  • ⁇ H is the enthalpy of unfolding at the midpoint of the thermal denaturation curve
  • ⁇ S is the entropy of unfolding at the midpoint of the thermal denaturation curve.
  • the a ⁇ umption ⁇ that were made in using the equation above are as follows: thermal melting curves are two state, described by an equilibrium between unfolded and folded peptide; and the enthalpy and entropy of unfolding are independent of temperature.
  • the T was also determined by taking the first derivative of the CD signal ( ⁇ ) with respect to temperature '1 (temperature in K) and finding the minimum of this function (Cantor, C.R. and P.R. Schimmel, Bio ⁇ physical Chemistry. W.H. Freeman, New York (1980)). All reported values of T m are those determined from curve fitting.
  • the error in the measurement of T is ⁇ 2*C except in case ⁇ in which 20'OT,, > 80*C, where the error is ⁇ 5°C.
  • T m The determinations of T m by d ⁇ /d(l/T) and by curve fitting agree to within the estimated errors. Additionally, the T m for each disulfide-bonded dimer was measured as a function of peptide concentration (over at least a 2.5-fold range of peptide concentration in the low micromolar range, as estimated by the ratio of the CD signal at low and high concentration) to determine if the dimers were associating to higher order oligomers (O'Shea, E.K. et al.. Science 243:538-542 (1989); O'Shea, E.K., et al. , Science 245:646-648 (1989)).
  • the disulfide-bonded heterodimer was incubated in redox buffer consisting of 100-500 ⁇ M reduced glutathione (Sigma) , 100-5— uM oxidized glutathione (Sigma) , 50 mM NaCl, 10 mM NaP0 4 (pH 7.4) at -23*C in an anaerobic cham ⁇ ber (Coy Laboratory Products, Inc.). Reactions were equilibrated at a total peptide concentration of -10-50 ⁇ M for 6-16 hr and quenched under anaerobic conditions with concentrated formic acid to a final concentration of 5% by volume (pH ⁇ 2) .
  • reaction products were analyzed by microbore HPLC (Waters, Inc.) using a linear-acetonitrile- H 2 0 gradient with segments of 0.1% to 0.25% increase in buffer B per minute.
  • Analytical Vydac C-18 column (0.46 x 25 cm) was used at a column temperature of 25 * C, 40 ⁇ C or 50 ⁇ C with a flow rate of 0.2 ml/ in.
  • Relative concentra ⁇ tions of the disulfide-bonded hetero- and homodimer ⁇ were determined by integration of the corresponding peaks (absorbance at 229 nm was monitored) .
  • Each redox reaction was determined to be at equilibrium by repeating the reaction using an equimolar mixture of reduced peptide ⁇ a ⁇ the ⁇ tarting material.
  • the value ⁇ for ⁇ Gs_pattyc. obtained from the ⁇ e two different ⁇ tarting points agreed to within 0.1 kcal/mol.
  • Residues at positions a and d comprise the 4-3 hydrophobic repeat characteristic of coiled coils, and residue ⁇ at positions e and g are predominantly charged amino acids that can be involved in intra- or interhelical electrostatic interactions (Hodges, R.S. e£ al. r Cold Spring Harbor Symp. Quant. Biol. f 37:299-310 (1972); McLachlan, A.D. and M. Stewart fJ. Mol. Biol. 98:293-304 (1975)) .
  • the Fos leucine zipper is very acidic, with a high concentration of acidic amino acids at position ⁇ e, g and b.
  • each chain has a large net negative charge at neutral pH (each chain has a net charge of -5) , one ex ⁇ pects that dimer formation would be disfavored due to general electrostatic repulsion between monomers.
  • the large increase in stability of the Fos homodimer upon titration to low pH can, thus, be explained to result from the relief of destabilizing intra- and interhelical elec ⁇ trostatic interactions between acidic residues close to the hydrophobic interface of the dimer.
  • the Jun leucine zipper has a slight net positive charge at neutral pH (dimer has a net charge of +2 at pH 7) ; in addition, the concentration of charge in the Jun leucine zipper is more spread out than in Fos. These properties are consistent with the les ⁇ dramatic increase in stability of the Jun homodimer at high pH. Qualita- tively, the pH dependence of stability for the Fos-Jun heterodimer changes as expected from an average of the pH dependence ⁇ for the homodimers. This result suggests that the Fos-Jun leucine zipper lack ⁇ dominant stabilizing electrostatic interactions that are unique to the hetero- dimer; in such a ca ⁇ e, a bellshaped pH dependence curve would be expected.
  • the boundary between the Fo ⁇ or Jun sequence and the GCN4 sequence was set by dividing the helical wheel diagram into two groups of residue ⁇ : the "in ⁇ ide” group, con ⁇ isting of the predominantly charged residue ⁇ at po ⁇ ition ⁇ e and g, and the "outside” group, consisting of residue ⁇ from po ⁇ ition ⁇ b, c and f.
  • Two sets of hybrid leucine zipper peptides were constructed.
  • One set of peptides has native sequence (N) from Fos or Jun at the inside position ⁇ and outside se ⁇ quence from GCN4; these peptides are referred to as N in .
  • the other set of peptides contains GCN4 sequence inside and Fos and Jun sequence outside; these peptides are referred to a ⁇ N ( ⁇ Jt .
  • heterodimer formation was quantit- ated from a redox experiment in which an equimolar mixture of the cysteine-containing Fos and Jun peptides is equili ⁇ brated in a redox buffer that facilitates disulfide bond formation.
  • K redox is determined from the ration of di ⁇ sulfide-bonded heterodimer to homodimers.
  • the free energy of specificity for heterodimer formation ( ⁇ G ) is equal to -RTlnK red0X + RTln2.
  • the N in peptides also form heterodimers preferential ⁇ ly, but with reduced specificity ( ⁇ G spec i ⁇ -1.2 kcal/mol).
  • the decrea ⁇ e in specificity of the N jn peptides appears to ari ⁇ e from a decrease in stability of the N in heterodimer; the N ; i hingen heterodimer is les ⁇ stable than the native hetero- dimer but the stabilities of the N in homodimer ⁇ are the same as the corresponding native homodimers (Table 1) .
  • the ⁇ peptides show essentially no specificity ( ⁇ G ⁇ pec is -0.1 kcal/mol).
  • the N out het ⁇ erodimer has a stability that i ⁇ intermediate between that of the two N out homodimers (Table 1) . Therefore, the inside residue ⁇ of the Fo ⁇ and Jun leucine zipper are nece ⁇ sary and sufficient to mediate preferential hetero ⁇ dimer formation.
  • N jn peptides show pH-dependent stability that closely resembles that of the native Fo ⁇ and Jun peptide ⁇ .
  • the pH dependence for each of the ⁇ dimer ⁇ doe ⁇ not resemble that of the corresponding native dimer, but resembles that of the GCN4 leucine zipper peptide. Therefore, residues at the inside positions (positions a, d, e and g) are al ⁇ o largely re ⁇ ponsible for the pH-depen- dent stability observed with the Fos and Jun peptide dimers.
  • ⁇ T T m (heterodimer AB) - 1/2 [T m (homodimer AA) + T m (homodimer BB) ]
  • ⁇ T appear ⁇ to be a quantitative measure of specificity, as it is linearly related to the free energy of specificity, ⁇ G ⁇ pec , with a *. proportionality constant of 7.4'C/kcal.
  • the hetero- dimeric peptide pairs can be grouped into three classes: specificity, antispecificity and additive.
  • the specifici ⁇ ty classes includes peptides pairs with positive, nonad- ditive differen ⁇ e ⁇ in T m ( T m ⁇ +8'C); the anti ⁇ pecificity class contains peptide pairs with negative, nonadditive differences in T m HI ( ⁇ ⁇ T perennial ⁇ -8'C)'; and the additive class consi ⁇ t ⁇ of peptide pair ⁇ in which the stability of the heterodimer is intermediate between that of the homodimers (+8°C > ⁇ T m -8 ⁇ C) .
  • the inside residues consist of the predominantly hydrophobic positions (a and d) and predominantly charged positions (e and g) .
  • GCN4-based hybrid peptides contain ⁇ ing native Fos and Jun ⁇ equence at the hydrophobic posi ⁇ tions (N ad ) or charged position ⁇ (N e g ) were made to evalu ⁇ ate the contribution of these groups of residue ⁇ to ⁇ peci- ficity.
  • the N e g peptides form heterodimer ⁇ with specific- ity ( ⁇ T m and AG ⁇ ,.) and stability (T at least as great as that of the native sequences (Table 1) .
  • the - N a . d peptides are slightly anti-specific.
  • the coupling of the ionization state of residue ⁇ at positions e and g to the stability of the Fos and Jun leucine zippers can be rationalized by using the crystal ⁇ tructure of a peptide corresponding to the GCN4 leucine zipper (O'Shea, E.K. et al.. Science 254:539-544 (1991)).
  • the methylene groups of the predominantly charged residues at positions e and g pack against the predominantly hydrophobic.residues at posi ⁇ tions a and d.
  • the hydrophobic interface is actual- ly formed by side chains from 4 residues of the heptad repeat.
  • terminal charged groups of re ⁇ idue ⁇ at positions e and g of the preceding heptad are close to each other. It i ⁇ likely that the close proximity of negatively charged residue ⁇ at po ⁇ itions e and g of oppos- ing Fos monomer ⁇ would di ⁇ rupt the complementary packing ⁇ een at the dimer interface of the coiled coil, accounting for the in ⁇ tability of the Fo ⁇ homodimer at neutral pH.
  • Example 3 Characterization of Homodimer ⁇ fACID-pl/ACID-pl and BASE-pl/BASE-Pl) and Heterodimer ⁇ fACID-Pl/BASE-Pl CD ⁇ tudie ⁇ were performed with a 1 mm, 0.5 cm or 1 mm cuvette on an Aviv Model 60DS or 62DS CD spectrophotometer equipped with a thermoelecric controller. All CD studies were done in the presence of pho ⁇ phate-buffered ⁇ aline (PBS: 150mM NaCl, 10 mM Na pho ⁇ phate, pH 7.0). All pep ⁇ tide concentrations were determined by absorbance at 280 nm in 6 M GuHCl (26) .
  • Thermal melting curve ⁇ were deter ⁇ mined by monitoring the CD ⁇ ignal at 222 nm a ⁇ a function of temperature. Melting temperature ⁇ were e ⁇ timated by taking the fir ⁇ t derivative of the CD ⁇ ignal with re ⁇ pect to temperature "1 (temperature in K) and finding the mini ⁇ mum of thi ⁇ function (35) . Rever ⁇ ibility wa ⁇ checked for all thermal melt ⁇ and, in general, melting curve ⁇ per- formed at pH ⁇ 8 are rever ⁇ ible (>90% recovery of ⁇ tarting CD ⁇ ignal) .
  • the ACID-pl/BASE-pl Heterodimer is Helical and Stable
  • PBS phosphate-buffered saline
  • the characteristic the helical minima at 222 and 208 nm in the spectrum of an equi olar mixture of ACID-pl and BASE-pl indicate that the mixture i ⁇ highly helical.
  • the helical ⁇ tructure formed by the ACID-pl and BASE-pl mixture i ⁇ ⁇ table, a ⁇ it undergoes a cooperative unfolding transition when denatured with urea.
  • the isolated peptides ⁇ how no evidence for cooperative unfold ⁇ ing.
  • Sedimentation equilibrium studies of ACID-pl and BASE-pl at 20"C in PBS, pH 7 indicate that this mixture is heterodimeric. Thu ⁇ , the ACID-pl and BASE-pl peptides as ⁇ ociate preferentially and fold a ⁇ a stable, helical heterodimer.
  • version ⁇ of the ACID and BASE peptide ⁇ (ACID-pln and BASE-pln) containing an N- terminal cy ⁇ teine followed by two glycine ⁇ were ⁇ ynthe- ⁇ ized (glycines were added to allow disulfide bond for ⁇ mation without the distortion of the coiled-coil ⁇ truc- ture.)
  • CD ⁇ tudie ⁇ demon ⁇ trate that the di ⁇ ulfide-bonded heterodimer and homodimer ⁇ are > 80% helical at 0°C and that each dimer ⁇ how ⁇ a cooperative thermal uinfolding tran ⁇ ition indicating that the ACID and BASE peptides can be stabilized sufficiently with a disulfide bond to permit folding as stable, helical dimers.
  • the concentration dependence of stability was ⁇ tudied for each of the di ⁇ ulfide-bonded peptide dimers to deter ⁇ mine if the orientation of the helices is parallel and if the dimers a ⁇ ociate to a higher-order oligomers.
  • the stabi;lity of each dimer is independent of peptide concentration over a range of concentration from -2.5 uM to 170 uM.
  • a heterodimer disulfide-bonded in the antiparallel prientat- ion ha ⁇ a CD ⁇ ignal that is dependent on peptide con ⁇ centration, indicating that the antiparallel dimers are a ⁇ ociating to higher-order oligomers.
  • our design strategy sought to drive preferential heterodimer formation by destabilizing the homodimer ⁇ rather than by ⁇ tabilizing the heterodimer. If eletro- ⁇ tatic destabilization of the homodimers is occurring, the homodimers will be unstable in conditions of neutral pH and low ionic strength and will become more stable when the charges are titrated at extremes of pH or when the ionic strength i ⁇ increa ⁇ ed. In contra ⁇ t, if stabili ⁇ zation of the heterodimer by ion pairs is present, the heterodimer will be most stable at neutral pH and will become less stable as the charge on residues involved in ionic interactions is titrated at pH extremes.
  • the increased ⁇ tability of the disulfide-bonded species allowed us to investigate the pH and ionic ⁇ trength dependence of ⁇ tability of the homodiemr ⁇ .
  • the ⁇ tability of both disulfide-bonded homodimers is strongly pH and ionic strength dependent.
  • the ACID-plN homodimer is > 80% more stable at acidic pH than at pH 7, demon- ⁇ trating clearly that it i ⁇ destabilized by acidic resi ⁇ dues at neutral pH.
  • the BASE-plN homodimer is de ⁇ tabilized at neutral pH by positively charged basic residues, as it becomes -20°C more stable as the pH is changed from neutral to basic pH.
  • the di- ⁇ ulfide-bonded homodimers are stabilized by increasing ionic strength.
  • the longer lysine side- chain allows for more flexibility and solvation of the terminal charged group.
  • the idea that the length of sidechains at positions e and g is important in determining homodimer stability is supported by studies of BASE peptides containing the non-natural amino acids ornithine (three methylene group ⁇ ) or diaminobutyric acid (two methylene groups) . BASE homodimers containing these non-natural amino acids are predominantly unfolded and the stability of the corre ⁇ ponding ACID/BASE heterodimer decrea ⁇ es with the decreasing sidechain length.
  • the disulfide-bonded heterodimers formed between ACID-plN and DAB-plN or ORN-plN have T m ' ⁇ of -63"C ad -80'C, respectively (the ACID-plN/BASE-pOlN heterodimer has a T B >100*C) .
  • the stability of the heterodimer is relatively independent of ionic ⁇ trength and pH.
  • the ratio of heterodimer to homodimer cannot be measured readily from an equilibrium mixture of the two peptide ⁇ .
  • the degree of ⁇ pecificity can be e ⁇ timated becau ⁇ e Ks._prajec (the equilibrium con ⁇ tant de ⁇ cribing the ratio of heterodimer to homodimer) i ⁇ linked thermodynamically to the di ⁇ ociation con ⁇ tant ⁇ for each of the dimer ⁇ . Therefore, the dissociation constants for each dimer were determined so that the degree of specificity, AG ⁇ pec (*-RTlnK ⁇ pec ) , could be esti ⁇ mated.
  • the following quantitation of specificity should be considered an estimate becau ⁇ e the linkage relation ⁇ hip relating the di ⁇ sociation constant ⁇ relie ⁇ upon the as- ⁇ umption that the monomer-dimer equilibria are two- ⁇ tate.
  • the di ⁇ ociation con ⁇ tant for the ACID-plN and BASE- plN heterodimer wa ⁇ determined by monitoring the CD ⁇ ignal a ⁇ a function of urea concentration.
  • the ⁇ e re ⁇ ulting denaturation curve wa ⁇ fit to a two-state model for mono ⁇ mer-dimer equilibrium to obtain a dis ⁇ ociation con ⁇ tant for the heterodimer of 3 x 10 "8 M.
  • Specificity can al ⁇ o be e ⁇ timated by mea ⁇ uring the difference between the melting temperature (T m ) of the heterodimer and the average of the T m s (melting tempera ⁇ ture) for the homodimer ⁇ ( ⁇ T m ) .
  • T m ha ⁇ been ea ⁇ ured for other di ⁇ ulfide-bonded leucine zipper peptide ⁇ and ha ⁇ been ⁇ hown to be related to ⁇ G by a proportionality con ⁇ tant of 7.4°C/kcal mol "1 .
  • this lower limit for ⁇ T implies that ⁇ G is at least -7.5 kcal/mol (>10 5 - 6 -fold preference for the heterodimer) .
  • the heterodimer is preferred over the ACID-pl and BASE-pl homodimers by at least -10 5 -fold. This degree of specificity is much greater than that observed for the Fos and Jun peptides where the Fos/Jun heterodimer is preferred by only -10 2 -fold.

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Abstract

Paires de peptides synthétiques conçues de sorte qu'ils s'apparient de manière spécifique l'un avec l'autre pour former un hétérodimère, après quoi ils se replient de préférence comme un hétérodimère hélicoïdal. Les membres peptides de la paire peuvent avoir une longueur quelconque, à condition qu'ils soient suffisamment longs pour être stables sous la forme hétérodimère et qu'ils puissent se replier en une configuration hélicoïdale. Les paires de peptides synthétiques ci-décrites peuvent être utilisées comme réactifs d'affinité pour isoler des molécules associées. En outre, étant donné que l'hétérodimère est formé de manière préférentielle et qu'il est très stable dans des conditions physiologiques, les peptides synthétiques ci-décrits sont indiqués pour une utilisation in vivo. Les paires de peptides synthétiques peuvent être utilisées, par exemple, dans des applications in vivo dans lesquelles deux molécules ou composants nécessaires pour un évènement donné doivent être mis en contact pour que l'évènement se produise. Les paires de peptides synthétiques ci-décrites permettent la mise en contact des composants avec une grande spécificité et une grande affinité. Dans une variante, les paires de peptides synthétiques peuvent être conçues de manière à empêcher la liaison ou l'interaction de molécules (par exemple, l'ADN et sa protéine de liaison) nécessaires pour qu'un évènement se produise. Les paires de peptides synthétiques peuvent également être utilisées comme fermeture moléculaire biodégradable, par exemple dans le cas de greffes et de sutures artificielles.
PCT/US1993/000884 1992-01-31 1993-02-01 Sequences d'acides amines s'appariant de maniere specifique WO1993015110A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031480A1 (fr) * 1994-05-18 1995-11-23 S.P.I. Synthetic Peptides Incorporated Composition de support d'immunogene de polypetide d'heterodimere et son procede d'utilisation
US5824483A (en) * 1994-05-18 1998-10-20 Pence Inc. Conformationally-restricted combinatiorial library composition and method
US6165335A (en) * 1996-04-25 2000-12-26 Pence And Mcgill University Biosensor device and method
US6787368B1 (en) 1999-03-02 2004-09-07 Helix Biopharma Corporation Biosensor method for detecting analytes in a liquid
US6872806B1 (en) 1999-06-25 2005-03-29 The Governors Of The University Of Alberta Polypeptide compositions formed using a coiled-coil template and methods of use

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010567A1 (fr) * 1990-12-14 1992-06-25 Creative Biomolecules, Inc. Polypeptide bioadhesif synthetique

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1992010567A1 (fr) * 1990-12-14 1992-06-25 Creative Biomolecules, Inc. Polypeptide bioadhesif synthetique

Non-Patent Citations (2)

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Title
JOURNAL OF BIOLOGICAL CHEMISTRY. (MICROFILMS) vol. 256, no. 3, 10 February 1981, BALTIMORE, MD US pages 1214 - 1224 R. S. HODGES ET AL. 'Synthetic Model for Two-stranded alpha-Helical Coiled-coils' cited in the application *
JOURNAL OF BIOLOGICAL CHEMISTRY. (MICROFILMS) vol. 259, no. 21, 10 November 1984, BALTIMORE, MD US pages 13253 - 13261 S.Y.M. LAU ET AL. 'Synthesis of a Model Protein of Defined Secondary and Quaternary Structure' cited in the application *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031480A1 (fr) * 1994-05-18 1995-11-23 S.P.I. Synthetic Peptides Incorporated Composition de support d'immunogene de polypetide d'heterodimere et son procede d'utilisation
US5824483A (en) * 1994-05-18 1998-10-20 Pence Inc. Conformationally-restricted combinatiorial library composition and method
AU708472B2 (en) * 1994-05-18 1999-08-05 S.P.I. Synthetic Peptides Incorporated Heterodimer polypeptide immunogen carrier composition and method
US6165335A (en) * 1996-04-25 2000-12-26 Pence And Mcgill University Biosensor device and method
US6461490B1 (en) 1996-04-25 2002-10-08 Pence, Inc. Biosensor device and method
US6478939B1 (en) 1996-04-25 2002-11-12 Pence, Inc. Biosensor device and method
US6787368B1 (en) 1999-03-02 2004-09-07 Helix Biopharma Corporation Biosensor method for detecting analytes in a liquid
US6872806B1 (en) 1999-06-25 2005-03-29 The Governors Of The University Of Alberta Polypeptide compositions formed using a coiled-coil template and methods of use
US7262272B2 (en) 1999-06-25 2007-08-28 The Governors Of The University Of Alberta Polypeptide compositions formed using a coiled-coil template and methods of use

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