WO2009049223A2

WO2009049223A2 - Methods and compositions for the site-selective incorporation of fluorinated amino acids into polypeptides

Info

Publication number: WO2009049223A2
Application number: PCT/US2008/079603
Authority: WO
Inventors: Susan E Cellitti; David H Jones; Youngha Ryu; Peter G. Schultz; Bernhard H. Geierstanger
Original assignee: Irm Llc; The Scripps Research Institute
Priority date: 2007-10-10
Filing date: 2008-10-10
Publication date: 2009-04-16
Also published as: WO2009049223A3

Abstract

Compositions including orthogonal aminoacyl-tRNA synthetases (O-RS) that preferentially aminoacylate an orthogonal tRNA (O-tRNA) with trifluoromethoxyphenylalanine are provided. Nucleic acids encoding these aminoacyl-tRNA synthetases are also provided.

Description

METHODS AND COMPOSITIONS FOR THE SITE-SELECTIVE INCORPORATION OF FLUORINATED AMINO ACIDS INTO

POLYPEPTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No.

60/979,036, filed October 10, 2007. This application also claims the benefit of U.S. Provisional Application No. 61/031,480, filed February 26, 2008. Both applications are incorporated herein by reference in their entireties for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under Grant GM62159 from the National Institutes of Health. The government may have certain rights to this invention.

FIELD OF THE INVENTION

[0003] This invention is in the field of translation biochemistry. The invention relates to compositions and methods of producing and/or analyzing site-specifically labeled proteins.

BACKGROUND OF THE INVENTION

[0004] Studies of biological macromolecules by NMR (Nuclear Magnetic Resonance) spectroscopy become increasingly difficult as the molecular weight of the molecule of interest increases, due to signal overlap and signal reduction resulting from faster transverse relaxation. Partial and uniform ²H-, ¹³C-, and ¹⁵N-labeling of proteins combined with heteronuclear, multidimensional NMR experiments can overcome these problems to some extent and has allowed the elucidation of structures of proteins with a molecular weight of 30 kDa (Goto and Kay (2000) Curr. Opin. Struct. Biol. 10:585; Gardner (1998) Annu. Rev. Biophys. Biomol. Struct. 27:357; Wuthrich (2003) Angew. Chem. Int. Ed. 42:3340; and Bax (1994) Curr. Opin. Struct. Biol. 4:738). The development of transverse relaxation optimized spectroscopy (TROSY) has extended the limit of solution NMR studies to systems as large as 900 kDa (Pervushin et al. (1997) Proc. Natl. Acad. ScL U.S.A. 94:12366; Fiaux et al. (2002) Nature 418:207; and Fernandez and Wider (2003) Curr. Opin. Struct. Biol. 13:570). Ultimately, however, the resonances in large proteins can become impossible to resolve even at the highest available magnetic fields.

[0005] Assignment of resonances to particular amino acids in a protein is a key step in

NMR studies. Such assignments can be facilitated, e.g., in studies of larger proteins, by site- specific labeling of one or more amino acids with an NMR active isotope (see, e.g., Ellman et al. (1992) J. Am. Chem. Soc. 114:7959).

[0006] To obtain sufficient quantities for NMR measurements, most isotopically labeled proteins are recombinantly expressed in E. coli using minimal media in combination with ¹³C glucose, ¹⁵N ammonium salts, and deuterium oxide. However, such techniques typically label many, if not all, amino acid residues in the protein simultaneously. Strategies for more selective incorporations of isotopes include feeding experiments with labeled amino acids in defined media (Gardner (1998) Annu. Rev. Biophys. Biomol. Struct. 27:357), often utilizing auxotrophic bacterial expression strains, using alpha-ketoisovalerate (Goto et al. (1999) J. Biomol. NMR 13:369) or pyruvate (Rosen et al. (1996) J. MoI. Biol. 263:627) for labeling of methyl groups in otherwise perdeuterated proteins, 'reverse isotope' labeling (Vuister et al. (1994) J. Am. Chem. Soc. 116:9206; Kelly et al. (1999) J. Biomol. NMR 14:79), segmental labeling by transsplicing (Yamazaki (1998) J. Am. Chem. Soc. 120:5591), or total and semi- synthesis by chemical ligation (Xu et al. (1999) Proc. Natl. Acad. ScL USA 96:388) and cell-free expression systems using chemically aminoacylated suppressor tRNAs (Yabuki et al. (1998) J. Biomol. NMR 11:295). Although site- specific incorporation of isotopic labels into a protein has been demonstrated by the latter method (Ellman et al. (1992) J. Am. Chem. Soc. 114:7959), the production of milligram quantities sufficient for NMR measurements is tedious and expensive. [0007] There is thus a need for compositions and methods that facilitate site-specific incorporation of isotopically labeled amino acids into proteins for NMR analysis. The present invention addresses these and other needs, as will be apparent upon review of the following disclosure.

SUMMARY OF THE INVENTION

[0008] The present invention provides compositions and methods for producing and/or analyzing spectroscopically labeled proteins through site-specific incorporation of spectroscopically labeled unnatural amino acids, such as a trifluoromethoxyphenylalanine into the proteins, using translation systems including orthogonal aminoacyl tRNA synthetases and orthogonal tRNAs. The invention also provides methods for producing orthogonal aminoacyl- tRNA synthetases that can be used in these translation systems. [0009] In one aspect, the invention provides a composition that includes an orthogonal aminoacyl-tRNA synthetase (O-RS) that preferentially aminoacylates an orthogonal tRNA (O- tRNA) with trifluoromethoxyphenylalanine (e.g., 2-amino-3-(4- (trifluoromethoxy)phenyl)propanoic acid (OCF₃Phe)). In some embodiments, the compositions of the invention also include one or more of: the trifluoromethoxyphenylalanine, a translation system, the O-tRNA (e.g., an O-tRNA that recognizes a selector codon, such as an amber codon), or a cell (e.g., an Escherichia coli (E. colϊ) cell). In addition to various aminoacyl-tRNA synthetases, the invention also provides nucleic acids encoding those enzymes. [0010] The nucleic acids and synthetases included in the different aspects of the invention have various embodiments. In some embodiments, for example, an O-RS is encoded by a nucleic acid that includes a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 2-15 and a polynucleotide sequence that hybridizes under highly stringent conditions over substantially an entire length of a nucleotide sequence of SEQ ID NOS: 2-15. In certain embodiments, the O-RS comprises an amino acid sequence of SEQ ID NO: 16 in which X₂₆ is K or I; X₃₂ is V, A, L, I, or H; X₆₄ is I or L; X₆₅ is A, G, L, H, P, S, T, or Q; X₇₀ is H or N; X_1Os is Q, K, A, W, E, T, Q, R, L, or H; X₁₀₉ is W, M, P, Q, A, G, or Y; X₁₅₅ is Q or S; X₁₅₈ is A, G, or S; X₁₅₉ is I, N, A, or V; and X₁₆₂ is K, V, Q, L, V, S, Y, or H. To further illustrate, the O-RS includes an amino acid sequence selected from the group consisting of: SEQ ID NOS: 17-30 in some embodiments of the invention. The O-RS described herein typically have improved K_m and/or K_cat for the trifluoromethoxyphenylalanine relative to a natural amino acid. [0011] In another aspect, the invention provides a method of producing a spectroscopically labeled protein. The method includes translating a nucleic acid that encodes a protein in a translation system to thereby produce the spectroscopically labeled protein. The nucleic acid includes a selector codon. In addition, the translation system includes an orthogonal tRNA (O-tRNA) that recognizes the selector codon, trifluoromethoxyphenylalanine (e.g., 2- amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF₃Phe)), and an orthogonal aminoacyl- tRNA synthetase (O-RS) that preferentially aminoacylates the O-tRNA with the trifluoromethoxyphenylalanine. The method also typically further includes analyzing the spectroscopically labeled protein, e.g., by subjecting the spectroscopically labeled protein to a spectroscopic technique, such as a nuclear magnetic resonance (NMR) technique. [0012] In another aspect, the invention provides a method of producing an orthogonal aminoacyl-tRNA synthetase (O-RS). The method includes (a) generating a library of variant aminoacyl-tRNA synthetase (RS) molecules derived from at least one RS, and (b) selecting or screening the library of variant RS molecules to identify one or more members that aminoacylate an orthogonal tRNA (O-tRNA) with trifluoromethoxyphenylalanine (e.g., 2-amino-3-(4- (trifluoromethoxy)phenyl)propanoic acid (OCF₃Phe)) to thereby produce the O-RS. [0013] In other exemplary aspects, the invention also provides kits that includes the orthogonal aminoacyl-tRNA synthetases, nucleic acids encoding those synthetases, or compositions described herein.

BRIEF DESCRIPTION OF THE DRAWINGS [0014] Figure 1 schematically depicts the structure of OCF₃Phe (2-amino-3-(4-

(trifluoromethoxy) phenyl) propanoic acid).

[0015] Figure 2 shows an ESI-MS spectra (panels A and B) of Z-domain expression with and without OCF₃Phe. More specifically, in panel A, an E. coli protein contaminating all samples is observed at 20,846 Da in the ESI-MS in the material produced with (panel A) and without (panel B) OCF₃Phe. The expected mass for Z-domain with OCF₃Phe incorporated is 7866 Da. As shown, the observed mass in panel (B) is 7865 Da.

[0016] Figure 3 shows a MALDI-TOF-MS-MS analysis after tryptic digestion of Z- domain with OCF₃Phe incorporated. In particular, the TOF-MS-MS analysis verifies incorporation of OCF₃Phe at the desired location in the tryptic peptide TSVDN(OCF₃Phe)INK (panel A). As shown in panel (B), only small amounts of the natural amino acids Tyr, Trp and Phe are misincorporated at the OCF₃Phe positions.

[0017] Figure 4 shows ¹⁹F-NMR spectra of FAS-TE mutants with OCF₃Phe incorporated.

For comparison a spectrum of a 0.5 mM sample of OCF₃Phe is shown. All samples (Table 3) in 20 mM deuterated imidazole (Cambridge Isotope Laboratories) pH 7.0, 100 mM NaCl, 0.5 mM TCEP (Pierce), 10% D₂O (Cambridge Isotope Laboratories). Spectra were recorded on an

Avance 400 MHz instrument (Bruker Biospin, Billerica, MA) equipped with a ¹HZ¹³CZ¹⁹FZ³¹P- QNP-cryoprobe at 300 K.

DETAILED DESCRIPTION

I. DEFINITIONS [0018] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular devices or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes combinations of two or more cells; reference to "a polynucleotide" includes, as a practical matter, many copies of that polynucleotide. [0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0020] As used herein, the term "orthogonal" refers to a molecule (e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl tRNA synthetase (0-RS)) that functions with endogenous components of a cell or other translation system with reduced efficiency as compared to a corresponding molecule that is endogenous to the cell or translation system, or that fails to function when paired with endogenous components of the cell or translation system. In the context of tRNAs and aminoacyl-tRNA synthetases, orthogonal refers to an inability or reduced efficiency (e.g., less than 20% efficiency, less than 10% efficiency, less than 5% efficiency, or less than 1% efficiency), of an orthogonal tRNA to function with an endogenous tRNA synthetase compared to the ability of an appropriate (e.g., homologous or analogous) endogenous tRNA to function when paired with the endogenous complementary tRNA synthetase; or of an orthogonal aminoacyl-tRNA synthetase to function with an endogenous tRNA as compared to the ability of an appropriate endogenous tRNA synthetase to function when paired with the endogenous complementary tRNA. The orthogonal molecule lacks a functionally normal naturally occurring endogenous complementary molecule in the cell or translation system. For example, an orthogonal tRNA in a cell is aminoacylated by any endogenous RS of the cell with reduced or even undetectable efficiency, when compared to aminoacylation of an endogenous tRNA by the endogenous RS. In another example, an orthogonal RS aminoacylates any endogenous tRNA in a cell of interest with reduced or even undetectable efficiency, as compared to aminoacylation of the endogenous tRNA by a complementary endogenous RS. A second orthogonal molecule can be introduced into the cell that functions when paired with the first orthogonal molecule. For example, an orthogonal tRNA/RS pair includes introduced complementary components that function together in the cell with an efficiency (e.g., 45% efficiency, 50% efficiency, 60% efficiency, 70% efficiency, 75% efficiency, 80% efficiency, 90% efficiency, 95% efficiency, or 99% or more efficiency) as compared to that of a control, e.g., a corresponding (e.g., analogous) tRNA/RS endogenous pair, or an active orthogonal pair (e.g., a tyrosyl or tryptophanyl orthogonal tRNA/RS pair). [0021] As used herein, an "orthogonal tRNA" (O-tRNA) is a tRNA that is orthogonal to a translation system of interest. The O-tRNA can exist charged with an amino acid, or in an uncharged state. It will be appreciated that an O-tRNA of the invention is advantageously used to insert essentially any amino acid, whether natural or unnatural, into a growing polypeptide, during translation, in response to a selector codon.

[0022] As used herein, an "orthogonal aminoacyl-tRNA synthetase" (O-RS) is an enzyme that preferentially aminoacylates an O-tRNA with an amino acid in a translation system of interest. [0023] As used herein, an "orthogonal tyrosyl-tRNA" (tyrosyl-O-tRNA) is a tRNA that is orthogonal to a translation system of interest, where the tRNA is: (1) identical or substantially similar to a naturally occurring tyrosyl-tRNA, (2) derived from a naturally occurring tyrosyl- tRNA by natural or artificial mutagenesis, (3) derived by any process that takes a sequence of a wild-type or mutant tyrosyl-tRNA sequence of (1) or (2) into account, or (4) homologous to a wild-type or mutant tyrosyl-tRNA. Exemplary tyrosyl-tRNAs are described in, e.g., Wang et al. (2001) Science 292:498 and U.S. patent application Ser. Nos. 10/126,927, 10/126,931,

10/825,867, and 60/634,151. The tyrosyl-tRNA can exist charged with an amino acid, or in an uncharged state. It is also to be understood that a "tyrosyl-O-tRNA" optionally is charged (aminoacylated) by a cognate synthetase with an amino acid other than tyrosine, e.g., with an unnatural amino acid. Indeed, it will be appreciated that a tyrosyl-O-tRNA of the invention is advantageously used to insert essentially any amino acid, whether natural or artificial, into a growing polypeptide, during translation, in response to a selector codon. [0024] As used herein, an "orthogonal tyrosyl aminoacyl synthetase" (tyrosyl-O-RS) is an enzyme that preferentially aminoacylates the tyrosyl-O-tRNA with an amino acid in a translation system of interest. The amino acid that the tyrosyl-O-RS loads onto the tyrosyl-O- tRNA can be any amino acid, whether natural, unnatural or artificial, and is not limited herein. The synthetase is optionally (1) the same as or homologous to a naturally occurring tyrosyl aminoacyl synthetase, (2) derived from a naturally occurring tyrosyl aminoacyl synthetase by natural or artificial mutagenesis, (3) derived by any process that takes a sequence of a wild-type or mutant tyrosyl aminoacyl synthetase sequence of (1) or (2) into account, or (4) homologous to a wild-type or mutant tyrosyl aminoacyl synthetase. Exemplary tyrosyl aminoacyl synthetases are described in, e.g., Wang et al. (2001) Science 292:498 and U.S. patent application Ser. Nos. 10/126,927, 10/126,931, 10/825,867, and 60/634,151. [0025] The term "cognate" refers to components that function together, e.g., an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase that preferentially aminoacylates the orthogonal tRNA. The components can also be referred to as being complementary. [0026] An O-RS "preferentially aminoacylates" a cognate O-tRNA when the O-RS charges the O-tRNA with an amino acid more efficiently than it charges any endogenous tRNA in an expression system. That is, when the O-tRNA and any given endogenous tRNA are present in a translation system in approximately equal molar ratios, the O-RS will charge the O-tRNA more frequently than it will charge the endogenous tRNA. Preferably, the relative ratio of O- tRNA charged by the O-RS to endogenous tRNA charged by the O-RS is high, preferably resulting in the O-RS charging the O-tRNA exclusively, or nearly exclusively, when the O-tRNA and endogenous tRNA are present in equal molar concentrations in the translation system. The relative ratio between O-tRNA and endogenous tRNA that is charged by the O-RS, when the O- tRNA and O-RS are present at equal molar concentrations, is greater than 1:1, preferably at least about 2: 1, more preferably 5:1, still more preferably 10:1, yet more preferably 20: 1, still more preferably 50:1, yet more preferably 75:1, and still more preferably 95:1, 98:1, 99:1, 100:1, 500:1, 1,000:1, 5,000:1 or higher.

[0027] The O-RS "preferentially aminoacylates an O-tRNA with an unnatural amino acid" when (a) the O-RS preferentially aminoacylates the O-tRNA compared to an endogenous tRNA, and when (b) that aminoacylation is specific for the unnatural amino acid, as compared to aminoacylation of the O-tRNA by the O-RS with any natural amino acid. That is, when the unnatural and natural amino acids are present in equal molar amounts in a translation system comprising the O-RS and O-tRNA, the O-RS will load the O-tRNA with the unnatural amino acid more frequently than with the natural amino acid. Preferably, the relative ratio of O-tRNA charged with the unnatural amino acid to O-tRNA charged with the natural amino acid is high. More preferably, O-RS charges the O-tRNA exclusively, or nearly exclusively, with the unnatural amino acid. The relative ratio between charging of the O-tRNA with the unnatural amino acid and charging of the O-tRNA with the natural amino acid, when both the natural and unnatural amino acids are present in the translation system in equal molar concentrations, is greater than 1:1, preferably at least about 2:1, more preferably 5:1, still more preferably 10:1, yet more preferably 20:1, still more preferably 50:1, yet more preferably 75:1, and still more preferably 95:1, 98:1, 99:1, 100:1, 500:1, 1,000:1, 5,000:1 or higher. [0028] The term "selector codon" refers to a codon recognized by the O-tRNA in the translation process and not typically recognized by an endogenous tRNA. The O-tRNA anticodon loop recognizes the selector codon on the mRNA and incorporates its amino acid, e.g., an unnatural amino acid, such as a spectroscopically labeled amino acid, at this site in the polypeptide. Selector codons can include, e.g., nonsense codons, such as stop codons (e.g., amber, ochre, and opal codons), four or more base codons, rare codons, codons derived from natural or unnatural base pairs, and/or the like.

[0029] The term "translation system" refers to the components that incorporate an amino acid into a growing polypeptide chain (protein). Components of a translation system can include, e.g., ribosomes, tRNAs, synthetases, mRNA and the like. The O-tRNA and/or the O-RSs of the invention can be added to or be part of an in vitro or in vivo translation system, e.g., in a non- eukaryotic cell, e.g., a bacterium (such as E. coli), or in a eukaryotic cell, e.g., a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, an insect cell, and/or the like. [0030] As used herein, the term "unnatural amino acid" refers to any amino acid, modified amino acid, and/or amino acid analog, such as a spectroscopically labeled amino acid (e.g., a trifluoromethoxyphenylalanine, etc.), that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine or pyrrolysine. [0031] As used herein, the term "trifluoromethoxyphenylalanine" refers to a trifluoromethoxy substituted phenylalanine amino acid. Typically, a trifluoromethoxy group is substituted at an ortho-, meta-, or para-position of the benzyl-group of the phenylalanine amino acid. In some embodiments, for example, a trifluoromethoxyphenylalanine is 2-amino-3-(4- (trifluoromethoxy)phenyl)propanoic acid (i.e., p-trifluoromethoxyphenylalanine; see, Figure 1). [0032] As used herein, the term "derived from" refers to a component that is isolated from or made using a specified molecule or organism, or information from the specified molecule or organism. For example, a polypeptide that is derived from a second polypeptide comprises an amino acid sequence that is identical or substantially similar to the amino acid sequence of the second polypeptide. In the case of polypeptides, the derived species can be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis or artificial random mutagenesis. The mutagenesis used to derive polypeptides can be intentionally directed or intentionally random. The mutagenesis of a polypeptide to create a different polypeptide derived from the first can be a random event (e.g., caused by polymerase infidelity) and the identification of the derived polypeptide can be serendipitous. Mutagenesis of a polypeptide typically entails manipulation of the polynucleotide that encodes the polypeptide. [0033] As used herein, the term "eukaryote" refers to organisms belonging to the

Kingdom Eukarya. Eukaryotes are generally distinguishable from prokaryotes by their typically multicellular organization (but not exclusively multicellular; for example, yeast), the presence of a membrane-bound nucleus and other membrane-bound organelles, linear genetic material (i.e., linear chromosomes), the absence of operons, the presence of introns, message capping and poly- A mRNA, and other biochemical characteristics, such as a distinguishing ribosomal structure. Eukaryotic organisms include, for example, animals (e.g., mammals, insects, reptiles, birds, etc.), ciliates, plants (e.g., monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc. [0034] As used herein, the term "prokaryote" refers to organisms belonging to the

Kingdom Monera (also termed Prokarya). Prokaryotic organisms are generally distinguishable from eukaryotes by their unicellular organization, asexual reproduction by budding or fission, the lack of a membrane-bound nucleus or other membrane-bound organelles, a circular chromosome, the presence of operons, the absence of introns, message capping and poly-A mRNA, and other biochemical characteristics, such as a distinguishing ribosomal structure. The Prokarya include subkingdoms Eubacteria and Archaea (sometimes termed "Archaebacteria"). Cyanobacteria (the blue green algae) and mycoplasma are sometimes given separate classifications under the Kingdom Monera. [0035] As used herein, the term "in response to" refers to the process in which a O-tRNA of the invention recognizes a selector codon and mediates the incorporation of the unnatural amino acid (e.g., the spectroscopically labeled unnatural amino acid), which is coupled to the tRNA, into the growing polypeptide chain.

[0036] As used herein, the term "encode" refers to any process whereby the information in a polymeric macromolecule or sequence string is used to direct the production of a second molecule or sequence string that is different from the first molecule or sequence string. As used herein, the term is used broadly, and can have a variety of applications. In one aspect, the term "encode" describes the process of semi-conservative DNA replication, where one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase. [0037] In another aspect, the term "encode" refers to any process whereby the information in one molecule is used to direct the production of a second molecule that has a different chemical nature from the first molecule. For example, a DNA molecule can encode an RNA molecule (e.g., by the process of transcription incorporating a DNA-dependent RNA polymerase enzyme). Also, an RNA molecule can encode a polypeptide, as in the process of translation. When used to describe the process of translation, the term "encode" also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, e.g., by the process of reverse transcription incorporating an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a polypeptide, where it is understood that "encode" as used in that case incorporates both the processes of transcription and translation.

[0038] A "polypeptide" (or a "protein") is a polymer comprising two or more amino acid residues. The polymer can additionally comprise non-amino acid elements such as labels, quenchers, blocking groups, or the like and can optionally comprise modifications such as glycosylation or the like. The amino acid residues of the polypeptide can be natural and/or unnatural and can be unsubstituted, unmodified, substituted or modified. [0039] A "spectroscopic label" is a moiety (e.g., an atom or a chemical group) whose presence in a protein can produce a measurable difference in a spectroscopic property of the protein, as compared to the corresponding protein lacking the spectroscopic label. For example, in an unnatural amino acid comprising a spectroscopic label, one or more atoms of the unnatural amino acid can be replaced by or substituted with the spectroscopic label (e.g., an atom can be replaced by an isotopic label or be substituted with a spin-label), or the spectroscopic label can be added to the unnatural amino acid (e.g., a fluorophore or a nitroxide radical spin-label can be covalently attached to the unnatural amino acid). A "spectroscopically labeled protein" comprising an unnatural amino acid with a spectroscopic label (e.g., attached either before or after incorporation of the unnatural amino acid into the protein) thus displays a measurable difference in at least one spectroscopic property as compared to the protein including the unnatural amino acid but lacking the spectroscopic label. [0040] In an unnatural amino acid that is "isotopically labeled", at least one atomic position in the amino acid is occupied exclusively or nearly exclusively by a single isotope of a given element, instead of being occupied by a mixture of the isotopes of that element at their natural abundance. The isotopic label can be the naturally most abundant isotope, or it can be a naturally less abundant isotope. Isotopic labels include, but are not limited to, NMR active isotopes and radioactive isotopes.

[0041] An "NMR active isotope" has a nonzero nuclear spin (e.g., a spin of Vi, 1, 3/2, 5/2, or 7/2). [0042] A "spin-label" is a paramagnetic moiety. Spin-labels typically comprise unpaired electrons.

[0043] The term "nucleic acid" or "polynucleotide" encompasses any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides (e.g., a typical DNA or RNA polymer), PNAs, modified oligonucleotides (e.g., oligonucleotides comprising nucleotides that are not typical to biological RNA or DNA, such as 2'-O-methylated oligonucleotides), and the like. A nucleic acid can be e.g., single- stranded or double-stranded. Unless otherwise indicated, a particular nucleic acid sequence of this invention optionally comprises or encodes complementary sequences, in addition to any sequence explicitly indicated.

[0044] A "polynucleotide sequence" or "nucleotide sequence" is a polymer of nucleotides

(an oligonucleotide, a DNA, a nucleic acid, etc.) or a character string representing a nucleotide polymer, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence (e.g., the complementary nucleic acid) can be determined.

[0045] As used herein, the term polynucleotide includes DNAs or RNAs that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein.

[0046] It will be appreciated that a great variety of modifications have been made to

DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.

[0047] The present application is directed to nucleic acid molecules at least 90%, 95%,

96%, 97%, 98% or 99% identical to the nucleic acid sequences disclosed herein irrespective of whether they encode a polypeptide having synthetase activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide synthetase functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having synthetase functional activity include, inter alia, (1) isolating a gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of genes, as described in Verma, et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) northern blot analysis for detecting mRNA expression in specific tissues. [0048] A "variant" of a polynucleotide or polypeptide, as the term is used herein, includes polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. A polynucleotide variant is a polynucleotide that differs in nucleotide sequence from another, reference polynucleotide. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical. Changes in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide with the same amino acid sequence as the reference. Changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence.

[0049] Owing to the degeneracy of the genetic code, "silent substitutions" (i.e., substitutions in a nucleic acid sequence that do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence that encodes an amino acid. Similarly, "conservative amino acid substitutions," in which one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct. Such conservative variations (or conservative variants) of each disclosed sequence are a feature of the present invention. [0050] "Conservative variants" or "conservative variations" of a particular nucleic acid sequence refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. One of skill will recognize that individual substitutions, deletions or additions which alter, add, or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 2% or 1%) in an encoded sequence are

"conservatively modified variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. Thus, "conservative variants" or "conservative variations" of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 2% or 1%, of the amino acids of the polypeptide sequence with an amino acid of the same conservative substitution group. The addition of sequences that do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional sequence, is a conservative variation of the basic nucleic acid.

[0051] Conservative substitutions providing functionally similar amino acids are well known in the art, where one amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., aromatic side chains or positively charged side chains), and therefore does not substantially change the functional properties of the polypeptide molecule. [0052] Comparative hybridization can be used to identify nucleic acids of the invention, including conservative variations of nucleic acids of the invention, and this comparative hybridization method is one method of distinguishing nucleic acids of the invention. In addition, target nucleic acids which hybridize to the nucleic acids represented by SEQ ID NOS: 2-15 under high, ultra-high and ultra-ultra high stringency conditions are a feature of the invention. Examples of such nucleic acids include those with one or a few silent or conservative nucleic acid substitutions as compared to a given nucleic acid sequence.

[0053] A test nucleic acid is said to specifically hybridize to a probe nucleic acid when it hybridizes at least 1/2 as well to the probe as to the perfectly matched complementary target, i.e., with a signal to noise ratio at lest 1/2 as high as hybridization of the probe to the target under conditions in which the perfectly matched probe binds to the perfectly matched complementary target with a signal to noise ratio that is at least about 5 to 10 fold as high as that observed for hybridization to any of the unmatched target nucleic acids.

[0054] Nucleic acids "hybridize" when they associate, typically in solution. Nucleic acids hybridize due to a variety of well characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes part I chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," (Elsevier, New York), as well as in Ausubel, infra. Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford University Press, Oxford, England, (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes 2 IRL

Press at Oxford University Press, Oxford, England (Hames and Higgins 2) provide details on the synthesis, labeling, detection and quantification of DNA and RNA, including oligonucleotides. [0055] An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42°C, with the hybridization being carried out overnight. An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, infra for a description of SSC buffer). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal. An example low stringency wash is 2x SSC at 40°C for 15 minutes. In general, a signal to noise ratio of 5 fold (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. [0056] "Stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993), supra, and in Hames and Higgins, 1 and 2. Stringent hybridization and wash conditions can easily be determined empirically for any test nucleic acid. For example, in determining highly stringent hybridization and wash conditions, the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents such as formalin in the hybridization or wash), until a selected set of criteria are met. For example, the hybridization and wash conditions are gradually increased until a probe binds to a perfectly matched complementary target with a signal to noise ratio that is at least 5 fold as high as that observed for hybridization of the probe to an unmatched target.

[0057] "Very stringent" conditions are selected to be equal to the thermal melting point

(T_m) for a particular probe. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe. For the purposes of the present invention, generally, "highly stringent" hybridization and wash conditions are selected to be about 5°C lower than the T_m for the specific sequence at a defined ionic strength and pH. [0058] "Ultra high-stringency" hybridization and wash conditions are those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least 10 fold as high as that observed for hybridization to any of the unmatched target nucleic acids. A target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least 1/2 that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-high stringency conditions.

[0059] Similarly, even higher levels of stringency can be determined by gradually increasing the hybridization and/or wash conditions of the relevant hybridization assay. For example, those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least 10, 20, 50, 100, or 500 fold or more as high as that observed for hybridization to any of the unmatched target nucleic acids. A target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least 1/2 that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra- ultra-high stringency conditions.

[0060] Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

[0061] DNA sequences generated by sequencing reactions may contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence, for example, one base insertion or deletion in an open reading frame of over 1000 bases. Notwithstanding any errors in the sequence data disclosed herein, the principles of the invention will nevertheless be readily comprehended by one skilled in the art. [0062] Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence shown in the appended sequence listing, or fragments thereof, will encode polypeptides "having synthetase functional activity". In fact, because degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having synthetase functional activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).

[0063] The terms "identical" or percent "identity", in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms available to persons of skill or by visual inspection. [0064] The phrase "substantially identical", in the context of two nucleic acids or polypeptides (e.g., DNAs encoding an O-tRNA or O-RS, or the amino acid sequence of an O- RS) refers to two or more sequences or subsequences that have at least about 60%, about 80%, about 90-95%, about 98%, or about 99%, or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. Such "substantially identical" sequences are typically considered to be "homologous", without reference to actual ancestry. Preferably, the "substantial identity" exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably, the sequences are substantially identical over at least about 150 residues, or over the full length of the two sequences to be compared. [0065] For sequence comparison and percent identity determination, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) to the reference sequence, based on the designated program parameters.

[0066] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2, 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, MoI. Biol. 48, 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85, 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., USA), or by visual inspection. [0067] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. MoI. Biol. 215, 403-10 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the world wide web at ncbi.nlm.nih.gov). See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89, 10915 (1989). In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 90, 5873-787 (1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. [0068] A variety of additional terms are defined or otherwise characterized herein.

II. INTRODUCTION

[0069] NMR spectroscopy is an established and very powerful biophysical method to study the structure, dynamics and function of proteins (Wuthrich, K. "The Second Decade - into the Third Millennium" Nature Structural Biology 1998, 5:492-495; Mittermaier, A. et al. "New tools provide new insights in NMR studies of protein dynamics" Science 2006, 312:224-8, and Tugarinov, V. et al. "Nuclear magnetic resonance spectroscopy of high-molecular- weight proteins" Annual Review of Biochemistry 2004, 73: 107-146). In principle, molecular processes such as binding or structural rearrangements can be deciphered by NMR with atomic resolution. However, the complexity of NMR spectra of large proteins in practice often provides a formidable challenge. Simplification of NMR spectra by amino acid-type specific isotope labeling (Muchmore, D. C. et al. "Expression and nitrogen- 15 labeling of proteins for proton and nitrogen- 15 nuclear magnetic resonance" Methods in Enzymology 1989, 177:44-73) including labeling with fluorinated analogs of natural amino acids (Gerig, J. T. "Fluorine NMR of Proteins" Progress in Nuclear Magnetic Resonance Spectroscopy 1994, 26:293-370; Frieden, C. et al. "The preparation of 19F-labeled proteins for NMR studies" Methods in Enzymology 2004, 380:400-15; and Danielson, M. A. et al. "Use of F-19 NMR to Probe Protein Structure and

Conformational Changes" Annual Review of Biophysics & Biomolecular Structure 1996, 25:163- 195), or selective isotope labeling of methyl groups in perdeuterated proteins (Tugarinov et al. "Methyl groups as probes of structure and dynamics in NMR studies of high-molecular- weight proteins" Chembiochem 2005, 6:1567-77; and Gardner, K. H. et al. "Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR" Biochemistry 1997, 36:1389- 401) is very helpful for many applications such as functional and binding studies. Complete resonance assignments that associate each NMR signal to an atom of a particular protein residue are not always necessary in order to provide useful information. The above mentioned labeling strategies do not necessarily provide a solution to the "assignment problem": Time-consuming heteronuclear NMR assignment experiments or a series of mutations are generally required to associate a given NMR resonance signal with a specific amino acid residue among many residues of that type. [0070] Recently developed methods for the in vivo incorporation of unnatural amino acids into proteins (Wang, L. et al. "Expanding the genetic code" Annual Review of Biophysics & Biomolecular Structure 2006, 35:225-49; Ryu, Y. et al. "Efficient incorporation of unnatural amino acids into proteins in Escherichia coli" Nature Methods 2006, 3:263-5; Wang, L. et al. "A general approach for the generation of orthogonal tRNAs" Chemistry & Biology 2001, 8:883-90; Xie, J. et al. "A chemical toolkit for proteins— an expanded genetic code" Nature Reviews Molecular Cell Biology 2006, 7:775-82; Xie, J. et al. "Adding amino acids to the genetic repertoire" Current Opinion in Chemical Biology 2005, 9:548-54; Xie, J. et al. "An expanding genetic code" Methods 2005, 36:227-38; and Liu, W. et al. "Genetic incorporation of unnatural amino acids into proteins in mammalian cells" Nat Methods 2007, 4:239-244) provide an unique opportunity to address these problems. Using an orthogonal amber tRNA/tRNA synthetase pair that specifically aminoacylates an amber tRNA with the desired unnatural amino acid and incorporates it into a protein at the amber nonsense codon UAG, any desired protein residue can, at least in principle, be substituted in vivo by an NMR-active, labeled unnatural amino acid. It has been shown that protein amounts sufficient for NMR studies can be produced using ¹⁵N- labeled O-methyl-phenylalanine (OMePhe) (Deiters, A. et al. "Site- specific in vivo labeling of proteins for NMR studies" Chembiochem 2005, 6:55-8). Because the NMR-active unnatural amino acid is only incorporated at the desired location, the approach instantly provides an "assignment" for the NMR signal of the unnatural amino acid. Monitoring the chemical shift change of a single resonance opens an avenue for focused, site-directed screening for binders, reducing the number of screening hits that bind to protein pockets of little interest for drug development. Unnatural amino acids incorporated at different sites in multiple samples can also be used to triangulate binding of a small molecule or a biomacromolecule via NOE measurements greatly simplifying the analysis. [0071] Fluorine represents an attractive NMR label since ¹⁹F has a natural abundance of

100% and is second to only hydrogen in its intrinsic sensitivity. The chemical shift of fluorine is highly sensitive to its environment and has range of several hundred ppm compared to tens of ppm for hydrogen (Gerig, J. T. "Fluorine NMR of Proteins" Progress in Nuclear Magnetic Resonance Spectroscopy 1994, 26:293-370; Frieden, C. et al. "The preparation of 19F-labeled proteins for NMR studies" Methods in Enzymology 2004, 380:400-15; and Danielson, M. A. et al. "Use of F- 19 NMR to Probe Protein Structure and Conformational Changes" Annual Review of Biophysics & Biomolecular Structure 1996, 25:163-195). In addition, one of the most appealing features of ¹⁹F NMR for protein NMR is the complete absence of a natural background. Fluorinated unnatural amino acids are therefore an attractive choice, e.g., for site- specific labeling of a protein with an NMR probe.

[0072] Although, with few exceptions, the genetic codes of all known organisms encode the same twenty amino acids, all that is required to add a new amino acid to the repertoire of an organism is a unique tRNA/aminoacyl-tRNA synthetase pair, a source of the amino acid, and a unique selector codon that specifies the amino acid (Furter (1998) Protein ScL, 7:419-426). The amber nonsense codon, TAG, together with orthogonal M. jannaschii and E. coli tRNA/synthetase pairs can be used to genetically encode a variety of amino acids with novel properties in E. coli (Wang et al., (2000) J. Am. Chem. Soc, 122:5010-5011; Wang et al., (2001) Science, 292:498-500; Wang et al., (2003) Proc. Natl. Acad. Sci. U.S.A., 100:56-61; Chin et al., (2002) Proc. Natl. Acad. Sci. U.S.A., 99:11020-11024; Wang and Schultz (2002) Chem.

Commun. 1:1), and yeast (Chin and Schultz, (2002) ChemBioChem, 3:1135-1137; Chin et al. (2003) Science 301:964-967), respectively.

[0073] In order to add additional synthetic amino acids, such as spectroscopically labeled unnatural amino acids, to the genetic code, e.g., in vivo, orthogonal pairs of an aminoacyl-tRNA synthetase and a suitable tRNA are needed that can function efficiently in the translational machinery, but that are "orthogonal" to the translation system at issue, meaning that the pairs function independently of the synthetases and tRNAs endogenous to the translation system. Desired characteristics of an orthogonal pair include a tRNA that decodes or recognizes only a specific new codon, e.g., a selector codon, that is not decoded by any endogenous tRNA, and an aminoacyl-tRNA synthetase that preferentially aminoacylates (or charges) its cognate tRNA with only a specific non-natural amino acid. The O-tRNA is also desirably not aminoacylated by endogenous synthetases. For example, in E. coli, an orthogonal pair will include an aminoacyl- tRNA synthetase that does not cross-react with any of the endogenous tRNAs, e.g., of which there are 40 in E. coli, and an orthogonal tRNA that is not substantially aminoacylated by any of the endogenous synthetases, e.g., of which there are 21 in E. coli.

[0074] A number of such O-tRNA/O-RS pairs have been described, and others can be produced by one of skill in the art. Such O-tRNA/O-RS pairs can be used to incorporate a variety of different unnatural amino acids at specific sites in proteins of interest.

[0075] As noted, assignment of resonances to particular amino acids in protein NMR studies can be facilitated by site-specific labeling of one or more amino acids in the protein with an NMR active isotope. Site-specific, efficient incorporation of isotopically labeled unnatural amino acids into proteins can thus facilitate resonance assignment during NMR studies of proteins. For example, it can often be useful, e.g., in solution studies of protein-ligand interactions, protein conformational changes, or catalysis, to only assign the single residue(s) of an active site or a ligand binding site, using for example the SEA-TROSY experiment (Pellecchia et al. (2001) J. Am. Chem. Soc. 123:4633). Introducing one or several site-specific NMR labels at such locations can greatly simplify the assignment problem and can thus enable detailed NMR solution studies of even very large proteins. Similarly, site- specific introduction of one or more spin-labels or paramagnetic metals can facilitate NMR signal assignments. [0076] Site-specific spectroscopic labeling of proteins can also be advantageous for use of spectroscopic techniques other than NMR (e.g., EPR spectroscopy, X-ray spectroscopy, mass spectroscopy, fluorescence spectroscopy, or vibrational (e.g., infrared or Raman) spectroscopy). For example, isotopic labeling can facilitate identification of peptide fragments in mass spectroscopy, incorporation of a fluorophore-containing unnatural amino acid (e.g., fluorophore- labeled L-phenylalanine or fluorophore-labeled p-acetyl-L-phenylalanine) can facilitate fluorescence spectroscopy, and incorporation of a spin-labeled unnatural amino acid can facilitate EPR. [0077] Accordingly, one aspect of the invention provides compositions and methods for producing spectroscopically labeled proteins through site-specific incorporation of spectroscopically labeled unnatural amino acids (e.g., trifluoromethoxyphenylalanines, such as p- trifluoromethoxyphenylalanine, etc.) into the proteins, using translation systems including orthogonal aminoacyl tRNA synthetases and orthogonal tRNAs. The present invention provides compositions and methods for producing and/or analyzing spectroscopically labeled proteins through site-specific incorporation of spectroscopically labeled unnatural amino acids, such as a trifluoromethoxyphenylalanine into the proteins, using translation systems including orthogonal aminoacyl tRNA synthetases and orthogonal tRNAs. The invention also provides methods for producing orthogonal aminoacyl-tRNA synthetases that can be used in these translation systems. Various aspects of the invention are illustrated in the representative example provided below.

III. ORTHOGONAL TRNAS, ORTHOGONAL AMINOACYL-TRNA SYNTHETASES, AND PAIRS THEREOF [0078] Translation systems that are suitable for making proteins that include one or more unnatural amino acids are described, e.g., in International Publication Numbers WO 2002/086075, entitled "Methods and composition for the production of orthogonal tRNA- aminoacyl-tRNA synthetase pairs" and WO 2002/085923, entitled "In vivo incorporation of unnatural amino acids." In addition, see International Application Number PCT/US2004/011786, filed Apr. 16, 2004, entitled "Expanding the Eukaryotic Genetic Code". Each of these applications is incorporated herein by reference in its entirety. Such translation systems generally comprise cells (which can be non-eukaryotic cells such as E. coli or eukaryotic cells such as yeast) that include an orthogonal tRNA (O-tRNA), an orthogonal aminoacyl tRNA- synthetase (O-RS), and an unnatural amino acid (in the present invention, unnatural amino acids containing spectroscopic labels, e.g., isotopic labels, are examples of such unnatural amino acids), where the O-RS aminoacylates the O-tRNA with the unnatural amino acid.

[0079] In general, when an orthogonal pair (an O-tRNA, e.g., a suppressor tRNA, a frameshift tRNA, or the like, and an O-RS) recognizes a selector codon and loads an amino acid in response to the selector codon, the orthogonal pair is said to "suppress" the selector codon. That is, a selector codon that is not recognized by the translation system's (e.g., cell's) endogenous machinery is not ordinarily translated, which can result in blocking production of a polypeptide that would otherwise be translated from the nucleic acid. When an orthogonal pair is present, the O-RS aminoacylates the O-tRNA with an unnatural amino acid of interest, such as a spectroscopically labeled unnatural amino acid (e.g., a trifluoromethoxyphenylalanine, etc.). The translation system (e.g., cell) uses the 0-tRNA/O-RS pair to incorporate the unnatural amino acid into a growing polypeptide chain, e.g., via a nucleic acid that encodes a polypeptide (protein) of interest, where the nucleic acid comprises a selector codon that is recognized by the O-tRNA. [0080] In certain embodiments of the invention, the translation system comprises a cell that includes an orthogonal aminoacyl-tRNA synthetase (O-RS), an orthogonal tRNA (O-tRNA), a spectroscopically labeled unnatural amino acid (e.g., a trifluoromethoxyphenylalanine, such as p-trifluoromethoxyphenylalanine or the like), and a nucleic acid that encodes a protein of interest, where the nucleic acid comprises the selector codon that is recognized by the O-tRNA. The cell can be a prokaryotic cell (such as an E. coli cell) or a eukaryotic cell (such as a yeast or mammalian cell). Typically, the orthogonal pair and the cell are derived from different sources (e.g., the cell can comprise an E. coli cell and the O-tRNA and the O-RS an M. jannaschii tyrosyl tRNA/tRNA synthetase pair, or the cell can comprise a eukaryotic cell and the O-tRNA and O- RS a prokaryotic orthogonal tRNA/tRNA synthetase pair). The translation system can also be a cell-free system, e.g., any of a variety of commercially available "in vitro" transcription/translation systems in combination with an 0-tRNA/O-RS pair and an unnatural amino acid as described herein.

[0081] The cell or other translation system optionally includes multiple 0-tRNA/O-RS pairs, which allows incorporation of more than one unnatural amino acid, e.g., two different spectroscopically labeled unnatural amino acids (comprising the same or different types of spectroscopic labels, e.g., isotopes) or a spectroscopically labeled unnatural amino acid and a different type of unnatural amino acid. For example, the cell can further include an additional different 0-tRNA/O-RS pair and a second unnatural amino acid, where this additional O-tRNA recognizes a second selector codon and this additional O-RS preferentially aminoacylates the O- tRNA with the second unnatural amino acid. For example, a cell that includes an 0-tRNA/O-RS pair (where the O-tRNA recognizes, e.g., an amber selector codon) can further comprise a second orthogonal pair, where the second O-tRNA recognizes a different selector codon (e.g., an opal codon, four-base codon, or the like). Desirably, the different orthogonal pairs are derived from different sources, which can facilitate recognition of different selector codons. [0082] The O-tRNA and/or the O-RS can be naturally occurring or can be, e.g., derived by mutation of a naturally occurring tRNA and/or RS, e.g., by generating libraries of tRNAs and/or libraries of RSs, from any of a variety of organisms and/or by using any of a variety of available mutation strategies. For example, one strategy for producing an orthogonal tRNA/aminoacyl-tRNA synthetase pair involves importing a heterologous (to the host cell) tRNA/synthetase pair from, e.g., a source other than the host cell, or multiple sources, into the host cell. The properties of the heterologous synthetase candidate include, e.g., that it does not charge any host cell tRNA, and the properties of the heterologous tRNA candidate include, e.g., that it is not aminoacylated by any host cell synthetase. A second strategy for generating an orthogonal pair involves generating mutant libraries from which to screen and/or select an O- tRNA or O-RS. These strategies can also be combined.

ORTHOGONAL TRNA (O TRNA)

[0083] An orthogonal tRNA (O-tRNA) of use in the compositions described herein desirably mediates incorporation of an unnatural amino acid, such as a spectroscopically labeled unnatural amino acid, into a protein that is encoded by a nucleic acid that comprises a selector codon that is recognized by the O-tRNA, e.g., in vivo or in vitro. An O-tRNA can be provided to the translation system, e.g., a cell, as the O-tRNA or as a polynucleotide that encodes the O- tRNA or a portion thereof. [0084] Methods of producing a recombinant orthogonal tRNA (O-tRNA) have been described and can be found, e.g., in international patent applications WO 2002/086075, entitled "Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA- synthetase pairs," PCT/US2004/022187 entitled "Compositions of orthogonal lysyl-tRNA and aminoacyl- tRNA synthetase pairs and uses thereof," and U.S. Ser. Nos. 60/479,931 and 60/496,548 entitled "Expanding the Eukaryotic Genetic Code." See also Forster et al., (2003) "Programming peptidomimetic synthetases by translating genetic codes designed de novo" Proc. Natl. Acad. Sci. USA 100(11):6353-6357; and, Feng et al., (2003), "Expanding tRNA recognition of a tRNA synthetase by a single amino acid change" Proc. Natl. Acad. Sci. USA 100(10): 5676-5681, as well as other references herein.

ORTHOGONAL AMINOACYL-TRNA SYNTHETASE (O RS)

[0085] An O-RS of the invention preferentially aminoacylates an O-tRNA with a spectroscopically labeled unnatural amino acid, namely, a trifluoromethoxyphenylalanine in vitro or in vivo. An O-RS of the invention can be provided to the translation system, e.g., a cell, by a polypeptide that includes an O-RS and/or by a polynucleotide that encodes an O-RS or a portion thereof. Exemplary O-RS amino acid sequences of the invention are provided in SEQ ID NOS: 17-30, while exemplary nucleic acids encoding those O-RSs are provided in SEQ ID NOS:2-15.

[0086] General methods of producing O-RS, and altering the substrate specificity of the synthetase, have been described and can be found, e.g., in WO 2002/086075 entitled "Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs," and International Application Number PCT/US2004/011786, filed Apr. 16, 2004, and PCT/US2004/022187 entitled "Compositions of orthogonal lysyl-tRNA and aminoacyl-tRNA synthetase pairs and uses thereof", filed JuI. 7, 2004, as well as other references herein. Specific representative examples of methods of producing O-RS that preferentially aminoacylate an O- tRNA with trifluoromethoxyphenylalanine are provided below.

SOURCE AND HOST ORGANISMS

[0087] The translational components of the invention can be derived from non-eukaryotic organisms. For example, the orthogonal O-tRNA can be derived from a non-eukaryotic organism (or a combination of organisms), e.g., an archaebacterium, such as Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-I, Ar chaeo globus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, Methanococcus maripaludis, Methanopyrus kandleri, Methanosarcina mazei, Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium, or the like, or a eubacterium, such as Escherichia coli, Thermus thermophilus, Bacillus stearothermphilus, or the like, while the orthogonal O-RS can be derived from a non-eukaryotic organism (or a combination of organisms), e.g., an archaebacterium, such as Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-I, Ar chaeo globus fulgidus , Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, Methanococcus maripaludis, Methanopyrus kandleri, Methanosarcina mazei, Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium, or the like, or a eubacterium, such as Escherichia coli, Thermus thermophilus, Bacillus stearothermphilus, or the like. In one embodiment, eukaryotic sources, e.g., plants, algae, protists, fingi, yeasts, animals (e.g., mammals, insects, arthropods, etc.), or the like, can also be used as sources of O-tRNAs and O-RSs. [0088] The individual components of an 0-tRNA/O-RS pair can be derived from the same organism or different organisms. In one embodiment, the 0-tRNA/O-RS pair is from the same organism. Alternatively, the O-tRNA and the O-RS of the 0-tRNA/O-RS pair are from different organisms.

[0089] The O-tRNA, O-RS or 0-tRNA/O-RS pair can be selected or screened in vivo or in vitro and/or used in a cell, e.g., a prokaryotic (non-eukaryotic) cell or a eukaryotic cell, to produce a polypeptide with an unnatural amino acid of interest. A non-eukaryotic cell can be from any of a variety of sources, e.g., a eubacterium, such as Escherichia coli, Thermus thermophilus, Bacillus stearothermphilus, or the like, or an archaebacterium, such as Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-I, Ar chaeo globus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, Methanococcus maripaludis,

Methanopyrus kandleri, Methanosarcina mazei, Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium, or the like. A eukaryotic cell can be from any of a variety of sources, e.g., a plant (e.g., a complex plant such as a monocot or a dicot), an algae, a protist, a fungus, a yeast (e.g., Saccharomyces cerevisiae), an animal (e.g., a mammal, an insect, an arthropod, etc.), or the like. For example, suitable insect host cells include, but are not limited to, Lepidopteran, Spodoptera frugiperda, Bombyx mori, Heliothis virescens, Heliothis zea, Mamestra brassicas, Estigmene acrea, and Trichoplusia ni insect cells; exemplary insect cell lines include BT1-TN-5B1-4 (High Five), BTI-TN-MGl, Sf9, Sf21, TN-368, D.Mel-2, and Schneider S-2 cells, among many others. To express a protein incorporating an unnatural amino acid, such insect cells are optionally infected with a recombinant baculovirus vector encoding the protein and a selector codon. A variety of baculovirus expression systems are known in the art and/or are commercially available, e.g., BaculoDirect™ (Invitrogen, Carlsbad, Calif.) and BD BaculoGold™ Baculovirus Expression Vector System (BD Biosciences, San Jose, Calif.). Compositions of cells with translational components of the invention are also a feature of the invention. [0090] See also, International Application Number PCT/US2004/011786, filed Apr. 16,

2004, for screening O-tRNA and/or O-RS in one species for use in another species.

SELECTOR CODONS

[0091] Selector codons of use in the invention expand the genetic codon framework of the protein biosynthetic machinery. For example, a selector codon includes, e.g., a unique three base codon, a nonsense codon, such as a stop codon, e.g., an amber codon (UAG), or an opal codon (UGA), an unnatural codon, at least a four base codon (e.g., AGGA), a rare codon, or the like. A number of selector codons can be introduced into a desired gene, e.g., one or more, two or more, more than three, etc. By using different selector codons, multiple orthogonal tRNA/synthetase pairs can be used that allow the simultaneous site-specific incorporation of multiple different unnatural amino acids into the protein of interest, using these different selector codons. Similarly, more than one copy of a given selector codon can by introduced into a desired gene to allow the site- specific incorporation of a given unnatural amino acid at multiple sites (e.g., two or more, three or more, etc.) in the protein of interest.

[0092] Conventional site-directed mutagenesis can be used to introduce the selector codon at the site of interest in a nucleic acid encoding a polypeptide of interest. When the O-RS, O-tRNA and the nucleic acid that encodes a polypeptide of interest are combined, e.g., in vivo, the spectroscopically labeled unnatural amino acid is incorporated in response to the selector codon to give a polypeptide containing the spectroscopically labeled unnatural amino acid at the specified position. [0093] The incorporation of unnatural amino acids such as spectroscopically labeled unnatural amino acids in vivo can be done without significant perturbation of the host cell. For example, in non-eukaryotic cells, such as Escherichia coli, because the suppression efficiency of a stop selector codon, the UAG codon, depends upon the competition between the O-tRNA, e.g., the amber suppressor tRNA, and release factor 1 (RFl) (which binds to the UAG codon and initiates release of the growing peptide from the ribosome), the suppression efficiency can be modulated by, e.g., either increasing the expression level of O-tRNA, e.g., the suppressor tRNA, or using an RFl deficient strain. In eukaryotic cells, because the suppression efficiency for a UAG codon depends upon the competition between the O-tRNA, e.g., the amber suppressor tRNA, and a eukaryotic release factor (e.g., eRF) (which binds to a stop codon and initiates release of the growing peptide from the ribosome), the suppression efficiency can be modulated by, e.g., increasing the expression level of O-tRNA, e.g., the suppressor tRNA. In addition, additional compounds can also be present that modulate release factor action, e.g., reducing agents such as dithiothreitol (DTT). [0094] Unnatural amino acids, including, e.g., spectroscopically labeled unnatural amino acids, can also be encoded with rare codons. For example, when the arginine concentration in an in vitro protein synthesis reaction is reduced, the rare arginine codon, AGG, has proven to be efficient for insertion of Ala by a synthetic tRNA acylated with alanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNAArg, which exists as a minor species in Escherichia coli. In addition, some organisms do not use all triplet codons. An unas signed codon AGA in Micrococcus luteus has been utilized for insertion of amino acids in an in vitro transcription/translation extract. See, e.g., Kowal and Oliver, Nucl. Acid. Res., 25:4685 (1997). Components of the invention can be generated to use these rare codons in vivo. [0095] Selector codons can also comprise extended codons, e.g., four or more base codons, such as four, five, six or more base codons. Examples of four base codons include, e.g., AGGA, CUAG, UAGA, CCCU, and the like. Examples of five base codons include, e.g., AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, and the like. Methods of the invention can include using extended codons based on frameshift suppression. Four or more base codons can insert, e.g., one or multiple unnatural amino acids into the same protein. In other embodiments, the anticodon loops can decode, e.g., at least a four-base codon, at least a five -base codon, or at least a six-base codon or more. Since there are 256 possible four-base codons, multiple unnatural amino acids can be encoded in the same cell using a four or more base codon. See also, Anderson et al. (2002) "Exploring the Limits of Codon and Anticodon Size" Chemistry and Biology, 9:237-244; and, Magliery (2001) "Expanding the Genetic Code: Selection of Efficient Suppressors of Four-base Codons and Identification of ^vShifty^v Four-base Codons with a Library Approach in Escherichia coli" J. MoI. Biol. 307: 755-769. [0096] For example, four-base codons have been used to incorporate unnatural amino acids into proteins using in vitro biosynthetic methods. See, e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al., (1999) J. Am. Chem. Soc, 121:34. CGGG and AGGU were used to simultaneously incorporate 2-naphthylalanine and an NBD derivative of lysine into streptavidin in vitro with two chemically acylated frameshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am. Chem. Soc, 121:12194. In an in vivo study, Moore et al. examined the ability of tRNA¹⁶¹¹ derivatives with NCUA anticodons to suppress UAGN codons (N can be U, A, G, or C), and found that the quadruplet UAGA can be decoded by a tRNA^Leu with a UCUA anticodon with an efficiency of 13 to 26% with little decoding in the 0 or -1 frame. See Moore et al., (2000) J. MoI. Biol., 298:195. In one embodiment, extended codons based on rare codons or nonsense codons can be used in the invention, which can reduce mis sense readthrough and frameshift suppression at other unwanted sites.

[0097] For a given system, a selector codon can also include one of the natural three base codons, where the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes a system that is lacking a tRNA that recognizes the natural three base codon, and/or a system where the three base codon is a rare codon.

[0098] Selector codons optionally include unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125. Properties of third base pairs include stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by a polymerase, and the efficient continued primer extension after synthesis of the nascent unnatural base pair.

Descriptions of unnatural base pairs which can be adapted for methods and compositions of the invention include, e.g., Hirao, et al., (2002) "An unnatural base pair for incorporating amino acid analogues into protein" Nature Biotechnology, 20:177-182. See also Wu, Y., et al., (2002) J. Am. Chem. Soc. 124:14626-14630. Other relevant publications are listed below. [0099] For in vivo usage, the unnatural nucleoside is membrane permeable and is phosphorylated to form the corresponding triphosphate. In addition, the increased genetic information is stable and not destroyed by cellular enzymes. Previous efforts by Benner and others took advantage of hydrogen bonding patterns that are different from those in canonical Watson-Crick pairs, the most noteworthy example of which is the iso-C:iso-G pair. See, e.g., Switzer et al, (1989) J. Am. Chem. Soc, 111:8322; and Piccirilli et al, (1990) Nature, 343:33; Kool, (2000) Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to some degree with natural bases and cannot be enzymatically replicated. Kool and co-workers demonstrated that hydrophobic packing interactions between bases can replace hydrogen bonding to drive the formation of base pair. See Kool, (2000) Curr. Opin. Chem. Biol 4:602; and Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort to develop an unnatural base pair satisfying all the above requirements, Schultz, Romesberg and co-workers have systematically synthesized and studied a series of unnatural hydrophobic bases. A PICS:PICS self -pair is found to be more stable than natural base pairs, and can be efficiently incorporated into DNA by

Klenow fragment of Escherichia coli DNA polymerase I (KF) (See, e.g., McMinn et al., (1999) J. Am. Chem. Soc, 121:11586; and Ogawa et al., (2000) J. Am. Chem. Soc, 122:3274). A 3MN:3MN self-pair can be synthesized by KF with efficiency and selectivity sufficient for biological function (See, e.g., Ogawa et al., (2000) J. Am. Chem. Soc, 122:8803). However, both bases act as a chain terminator for further replication. A mutant DNA polymerase has been recently evolved that can be used to replicate the PICS self pair. In addition, a 7AI self pair can be replicated (See, e.g., Tae et al., (2001) J. Am. Chem. Soc, 123:7439). A novel metallobase pair, DipicPy, has also been developed, which forms a stable pair upon binding Cu(II) (See Meggers et al., (2000) J. Am. Chem. Soc, 122:10714). Because extended codons and unnatural codons are intrinsically orthogonal to natural codons, the methods of the invention can take advantage of this property to generate orthogonal tRNAs for them. [0100] A translational bypassing system can also be used to incorporate a spectroscopically labeled unnatural amino acid or other unnatural amino acid into a desired polypeptide. In a translational bypassing system, a large sequence is inserted into a gene but is not translated into protein. The sequence contains a structure that serves as a cue to induce the ribosome to hop over the sequence and resume translation downstream of the insertion.

CHEMICAL SYNTHESIS OF UNNATURAL AMINO ACIDS

[0101] Many different unnatural amino acids (e.g., trifluoromethoxyphenylalanines, such as p-trifluoromethoxyphenylalanine, etc.) are commercially available, e.g., from Sigma (USA) or Aldrich (Milwaukee, Wis., USA). Those spectroscopically labeled unnatural amino acids that are not commercially available are optionally synthesized as provided in various publications or using standard methods known to those of skill in the art. For organic synthesis techniques, see, e.g., Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). Additional publications describing the synthesis of unnatural amino acids include, e.g., WO 2002/085923 entitled "In vivo incorporation of Unnatural Amino Acids;" Matsoukas et al. (1995) J. Med. Chem. 38:4660-4669; King and Kidd (1949) "A New Synthesis of Glutamine and of γ-Dipeptides of Glutamic Acid from Phthylated Intermediates" J. Chem. Soc. 3315-3319; Friedman and Chatterrji (1959) "Synthesis of Derivatives of Glutamine as Model Substrates for Anti-Tumor Agents" J. Am. Chem. Soc. 81:3750-3752; Craig et al. (1988) "Absolute Configuration of the Enantiomers of 7-Chloro-4 [[4- (diethylamino)-l-methylbutyl]amino]quinoline (Chloroquine)" J. Org. Chem. 53:1167-1170; Azoulay et al. (1991) "Glutamine analogues as Potential Antimalarials" Eur. J. Med. Chem. 26:201-205; Koskinen and Rapoport (1989) "Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino Acid Analogues" J. Org. Chem. 54:1859-1866; Christie and Rapoport (1985) "Synthesis of Optically Pure Pipecolates from L-Asparagine: Application to the Total Synthesis of (+)-Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization" J. Org. Chem. 1989:1859-1866; Barton et al. (1987) "Synthesis of Novel α-Amino- Acids and Derivatives Using Radical Chemistry: Synthesis of L- and D- α- Amino- Adipic Acids, L-α-aminopimelic Acid and Appropriate Unsaturated Derivatives" Tetrahedron Lett. 43:4297- 4308; and, Subasinghe et al. (1992) "Quisqualic acid analogues: synthesis of beta-heterocyclic 2- aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site" J. Med. Chem. 35:4602-4607 (See also International Application Number PCT/US03/41346, entitled "Protein Arrays," filed on Dec. 22, 2003).

CELLULAR UPTAKE OF UNNATURAL AMINO ACIDS

[0102] Unnatural amino acid (e.g., trifluoromethoxyphenylalanines) uptake by a cell is one issue that is typically considered when designing and selecting unnatural amino acids, e.g., for incorporation into a protein. For example, the high charge density of α-amino acids suggests that these compounds are unlikely to be cell permeable. Natural amino acids are taken up into the cell via a collection of protein-based transport systems often displaying varying degrees of amino acid specificity. A rapid screen can be done which assesses which unnatural amino acids, if any, are taken up by cells. See, e.g., toxicity assays in, e.g., International Application Number PCT/US03/41346, supra, and Liu and Schultz (1999) "Progress toward the evolution of an organism with an expanded genetic code" Proc. Natl. Acad. Sci. USA 96:4780-4785. Although uptake is easily analyzed with various assays, an alternative to designing unnatural amino acids that are amenable to cellular uptake pathways is to provide biosynthetic pathways to create amino acids in vivo.

BIOSYNTHESIS OF UNNATURAL AMINO ACIDS

[0103] Many biosynthetic pathways already exist in cells for the production of amino acids and other compounds. While a biosynthetic method for a particular unnatural amino acid may not exist in nature, e.g., in a cell, other approaches are known. For example, biosynthetic pathways for unnatural amino acids are optionally generated in host cell by adding new enzymes or modifying existing host cell pathways. Additional new enzymes are optionally naturally occurring enzymes or artificially evolved enzymes. For example, the biosynthesis of p- aminophenylalanine (as presented in an example in WO 2002/085923, supra) relies on the addition of a combination of known enzymes from other organisms. The genes for these enzymes can be introduced into a cell by transforming the cell with a plasmid comprising the genes. The genes, when expressed in the cell, provide an enzymatic pathway to synthesize the desired compound. Additional enzyme sequences are found, e.g., in Genbank. Artificially evolved enzymes are also optionally added into a cell in the same manner. In this manner, the cellular machinery and resources of a cell are manipulated to produce unnatural amino acids. [0104] Indeed, any of a variety of methods can be used for producing novel enzymes for use in biosynthetic pathways, or for evolution of existing pathways, for the production of unnatural amino acids, in vitro or in vivo. Many available methods of evolving enzymes and other biosynthetic pathway components can be applied to the present invention to produce unnatural amino acids (or, indeed, to evolve synthetases to have new substrate specificities or other activities of interest). For example, DNA shuffling is optionally used to develop novel enzymes and/or pathways of such enzymes for the production of unnatural amino acids (or production of new synthetases), in vitro or in vivo. See, e.g., Stemmer (1994) "Rapid evolution of a protein in vitro by DNA shuffling" Nature 370(4):389-391; and Stemmer (1994) "DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution" Proc. Natl. Acad. Sci. USA. 91:10747-10751. A related approach shuffles families of related (e.g., homologous) genes to quickly evolve enzymes with desired characteristics. An example of such a "family gene shuffling" method is found in Crameri et al. (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution" Nature

391(6664):288-291. New enzymes (whether biosynthetic pathway components or synthetases) can also be generated using a DNA recombination procedure known as "incremental truncation for the creation of hybrid enzymes" ("ITCHY"), e.g., as described in Ostermeier et al. (1999) "A combinatorial approach to hybrid enzymes independent of DNA homology" Nature Biotech 17:1205. This approach can also be used to generate a library of enzyme or other pathway variants which can serve as substrates for one or more in vitro or in vivo recombination methods. See, also, Ostermeier et al. (1999) "Combinatorial Protein Engineering by Incremental Truncation" Proc. Natl. Acad. Sci. USA 96: 3562-67, and Ostermeier et al. (1999) "Incremental Truncation as a Strategy in the Engineering of Novel Biocatalysts" Biological and Medicinal Chemistry 7: 2139-2144. Another approach uses exponential ensemble mutagenesis to produce libraries of enzyme or other pathway variants that are, e.g., selected for an ability to catalyze a biosynthetic reaction relevant to producing an unnatural amino acid (or a new synthetase). In this approach, small groups of residues in a sequence of interest are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Examples of such procedures, which can be adapted to the present invention to produce new enzymes for the production of unnatural amino acids (or new synthetases) are found in Delegrave and Youvan (1993) Biotechnology Research 11:1548-1552. In yet another approach, random or semi-random mutagenesis using doped or degenerate oligonucleotides for enzyme and/or pathway component engineering can be used, e.g., by using the general mutagenesis methods of e.g., Arkin and Youvan (1992) "Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis" Biotechnology 10:297-300; or Reidhaar-Olson et al. (1991) "Random mutagenesis of protein sequences using oligonucleotide cassettes" Methods Enzymol. 208:564- 86. Yet another approach, often termed a "non-stochastic" mutagenesis, which uses polynucleotide reassembly and site-saturation mutagenesis can be used to produce enzymes and/or pathway components, which can then be screened for an ability to perform one or more synthetase or biosynthetic pathway function (e.g., for the production of unnatural amino acids in vivo) (See, e.g., Short "Non-Stochastic Generation of Genetic Vaccines and Enzymes" WO 00/46344).

[0105] An alternative to such mutational methods involves recombining entire genomes of organisms and selecting resulting progeny for particular pathway functions (often referred to as "whole genome shuffling"). This approach can be applied to the present invention, e.g., by genomic recombination and selection of an organism (e.g., an E. coli or other cell) for an ability to produce an unnatural amino acid (or intermediate thereof). For example, methods taught in the following publications can be applied to pathway design for the evolution of existing and/or new pathways in cells to produce unnatural amino acids in vivo: Patnaik et al. (2002) "Genome shuffling of lactobacillus for improved acid tolerance" Nature Biotechnology, 20(7): 707-712; and Zhang et al. (2002) "Genome shuffling leads to rapid phenotypic improvement in bacteria" Nature 415: 644-646.

[0106] Other techniques for organism and metabolic pathway engineering, e.g., for the production of desired compounds are also available and can also be applied to the production of unnatural amino acids. Examples of publications teaching useful pathway engineering approaches include: Nakamura and White (2003) "Metabolic engineering for the microbial production of 1,3 propanediol" Curr. Opin. Biotechnol. 14(5):454-9; Berry et al. (2002) "Application of Metabolic Engineering to improve both the production and use of Biotech Indigo" J. Industrial Microbiology and Biotechnology 28:127-133; Banta et al. (2002) "Optimizing an artificial metabolic pathway: Engineering the cofactor specificity of

Corynebacterium 2,5-diketo-D-gluconic acid reductase for use in vitamin C biosynthesis" Biochemistry 41:6226-36; Selivonova et al. (2001) "Rapid Evolution of Novel Traits in Microorganisms" Applied and Environmental Microbiology 67:3645, and many others. [0107] Regardless of the method used, typically, the unnatural amino acid produced with an engineered biosynthetic pathway of the invention is produced in a concentration sufficient for efficient protein biosynthesis, e.g., a natural cellular amount, but not to such a degree as to significantly affect the concentration of other cellular amino acids or to exhaust cellular resources. Typical concentrations produced in vivo in this manner are about 0.05 mM to about 10 mM. Once a cell is engineered to produce enzymes desired for a specific pathway and an unnatural amino acid is generated, in vivo selections are optionally used to further optimize the production of the unnatural amino acid for both ribosomal protein synthesis and cell growth.

MUTAGENESIS AND OTHER MOLECULAR BIOLOGY TECHNIQUES

[0108] Polynucleotides and polypeptides of the invention and used in the invention can be manipulated using molecular biological techniques. General texts which describe molecular biological techniques include Berger and Kimmel, Guide to Molecular Cloning Techniques,

Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.; Sambrook et al., Molecular Cloning— A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005)). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics related to, e.g., the generation of nucleic acids including genes that include selector codons for production of proteins that include unnatural amino acids and to generation of orthogonal tRNAs, orthogonal synthetases, and pairs thereof. [0109] Various types of mutagenesis are optionally used in the invention, e.g., to insert selector codons that encode an unnatural amino acid in a protein of interest into a nucleic acid (e.g., into a DNA that encodes an RNA that is to be translated to produce the protein). They include, but are not limited to, site-directed mutagenesis, random point mutagenesis, homologous recombination, DNA shuffling or other recursive mutagenesis methods, chimeric construction, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like, or any combination thereof. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction- selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like.

[0110] Host cells are genetically engineered (e.g., transformed, transduced or transfected) with a relevant nucleic acid, e.g., a nucleic acid encoding an O-tRNA, O-RS, or a protein of interest including a selector codon, e.g., in a cloning vector or an expression vector. For example, the coding regions for the orthogonal tRNA, the orthogonal tRNA synthetase, and the protein to be derivatized are operably linked to gene expression control elements that are functional in the desired host cell. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and/or integration in prokaryotes, eukaryotes, or preferably both. See Giliman and Smith (1979) Gene 8:81; Roberts et al. (1987) Nature 328:731; Schneider et al. (1995) Protein Expr. Purif. 6435:10; Ausubel, Sambrook, Berger (all supra). The vector can be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide. The vectors are introduced into cells and/or microorganisms by standard methods including electroporation (From et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles or on the surface (Klein et al. (1987) Nature 327:70-73), and/or the like.

[0111] A catalog of bacteria and bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1996) Ghema et al. (eds.) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Sambrook (supra), Ausubel (supra), and in Watson et al. (1992) Recombinant DNA Second Edition, Scientific American Books (New York). In addition, essentially any nucleic acid (and virtually any labeled nucleic acid, whether standard or non-standard) can be custom or standard ordered from any of a variety of commercial sources, such as the Midland Certified Reagent Company (Midland, Tex.; available on the World Wide Web at mcrc.com), The Great American Gene Company (Ramona, Calif.; available on the World Wide Web at genco.com), ExpressGen Inc. (Chicago, 111.; available on the World Wide Web at expressgen.com), Operon Technologies Inc. (Alameda, Calif.) and many others. [0112] The engineered host cells can be cultured in conventional nutrient media modified as appropriate for such activities as, for example, screening steps, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic organisms. Other useful references, e.g. for cell isolation and culture (e.g., for subsequent nucleic acid isolation) include Freshney (2000) Culture of Animal Cells, a Manual of Basic Technique, fourth edition, Wiley-Liss, New York and the references cited therein; Higgins and Hames (eds) (1999) Protein Expression: A Practical Approach, Practical Approach Series, Oxford University Press; Shuler et al. (eds) (1994) Baculovirus Expression Systems and Biopesticides, Wiley-Liss; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N. Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FIa.

IV. METHODS FOR PRODUCING LABELED PROTEINS AND RESULTING COMPOSITIONS

[0113] As noted, one aspect of the invention provides methods for producing a spectroscopically labeled protein. One general class of embodiments provides methods in which a nucleic acid that encodes the protein is translated in a translation system. The nucleic acid includes a selector codon. The translation system includes an orthogonal tRNA (O-tRNA) that recognizes the selector codon, an unnatural amino acid comprising a spectroscopic label (e.g., trifluoromethoxyphenylalanines, such as p-trifluoromethoxyphenylalanine, etc.), and an orthogonal aminoacyl tRNA synthetase (O-RS) that preferentially aminoacylates the O-tRNA with the unnatural amino acid. Exemplary O-RS amino acid sequences of the invention are provided in SEQ ID NOS: 17-30, while exemplary nucleic acids encoding those O-RSs are provided in SEQ ID NOS:2-15. The unnatural amino acid is incorporated into the protein as it is translated in the translation system, thereby producing the spectroscopically labeled protein. Exemplary translation systems including 0-tRNA/O-RS pairs, exemplary selector codons, and exemplary unnatural amino acids have been described above.

[0114] Another general class of embodiments provides methods in which a nucleic acid that encodes the protein is translated in a translation system. The nucleic acid includes a selector codon for incorporating an unnatural amino acid at a specific position in the protein. The translation system includes an orthogonal tRNA (O-tRNA) that recognizes the selector codon, the unnatural amino acid, and an orthogonal aminoacyl tRNA synthetase (O-RS) that preferentially aminoacylates the O-tRNA with the unnatural amino acid (e.g., trifluoromethoxyphenylalanines, such as p-trifluoromethoxyphenylalanine, etc.). The unnatural amino acid is incorporated into the protein as it is translated, thereby producing a translated protein comprising the unnatural amino acid at the specific position. A spectroscopic label is attached (e.g., covalently attached) to the unnatural amino acid in the translated protein, thereby producing the spectroscopically labeled protein. The translated protein is optionally purified from the translation system prior to attachment of the spectroscopic label. Exemplary translation systems including 0-tRNA/O-RS pairs, exemplary selector codons, and exemplary unnatural amino acids have been described above.

[0115] It is worth noting that the methods for producing spectroscopically labeled proteins provide the ability to synthesize proteins that comprise spectroscopically labeled unnatural amino acids in large useful quantities. Thus, in one aspect, a composition is provided that includes, e.g., at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 50 milligrams, or at least 100 milligrams or more of a protein that comprises a spectroscopically labeled unnatural amino acid (or multiple unnatural amino acids), or an amount that can be achieved with in vivo protein production methods (details on recombinant protein production and purification are provided herein). In another aspect, the protein is optionally present in the composition at a concentration of, e.g., at least 10 micrograms of protein per liter, at least 50 micrograms of protein per liter, at least 75 micrograms of protein per liter, at least 100 micrograms of protein per liter, at least 200 micrograms of protein per liter, at least 250 micrograms of protein per liter, at least 500 micrograms of protein per liter, at least 1 milligram of protein per liter, or at least 10 milligrams of protein per liter or more, in, e.g., a cell lysate, a buffer, a pharmaceutical buffer, or other liquid suspension (e.g., in a volume of, e.g., anywhere from about 1 nL to about 100 L). The production of large quantities (e.g., greater that that typically possible with other methods, e.g., in vitro translation) of a protein in a cell including at least one spectroscopically labeled unnatural amino acid is a feature of the invention.

[0116] In one aspect of the invention, a composition includes at least one protein with at least one, and optionally, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more unnatural amino acids, e.g., spectroscopically labeled unnatural amino acids and/or other unnatural amino acids. The unnatural amino acids can be the same or different, e.g., there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnatural amino acids. In another aspect, a composition includes a protein with at least one, but fewer than all, of a particular amino acid present in the protein substituted with the spectroscopically labeled unnatural amino acid. For a given protein with more than one unnatural amino acid, the unnatural amino acids can be identical or different (e.g., the protein can include two or more different types of unnatural amino acids, or can include two of the same unnatural amino acid). For a given protein with more than two unnatural amino acids, the unnatural amino acids can be the same, different or a combination of a multiple unnatural amino acid of the same kind with at least one different unnatural amino acid.

[0117] Essentially any protein (or portion thereof) that includes an unnatural amino acid, or that encodes multiple different unnatural amino acids (and any corresponding coding nucleic acid, e.g., which includes one or more selector codons), can be produced using the compositions and methods herein. No attempt is made to identify the hundreds of thousands of known proteins, any of which can be modified to include one or more unnatural amino acid, e.g., by tailoring any available mutation methods to include one or more appropriate selector codon in a relevant translation system. Common sequence repositories for known proteins include GenBank EMBL, DDBJ and the NCBI. Other repositories can easily be identified by searching the internet.

[0118] Typically, the proteins are, e.g., at least 60%, at least 70%, at least 75%, at least

80%, at least 90%, at least 95%, or at least 99% or more identical to any available protein (e.g., a therapeutic protein, a diagnostic protein, an industrial enzyme, or a domain or other portion thereof, and the like), and they comprise one or more unnatural amino acid. Essentially any protein whose structure is of interest can be modified to include a spectroscopically labeled unnatural amino acid. Examples of therapeutic, diagnostic, and other proteins that can be modified to comprise one or more spectroscopically labeled unnatural amino acids can be found, but are not limited to, those in International Application Number PCT/US2004/011786, filed Apr. 16, 2004, entitled "Expanding the Eukaryotic Genetic Code;" and, WO 2002/085923, entitled "In vivo incorporation of unnatural amino acids." Examples of therapeutic, diagnostic, and other proteins that can be modified to comprise one or more spectroscopically labeled unnatural amino acids include, but are not limited to, e.g., Alpha- 1 antitrypsin, Angiostatin, Antihemolytic factor, antibodies (further details on antibodies are found below), Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, C--X--C chemokines (e.g., T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-IO, GCP-2, NAP-4, SDF-I, PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractant protein- 1, Monocyte chemoattractant protein-2, Monocyte chemoattractant protein-3, Monocyte inflammatory protein-1 alpha, Monocyte inflammatory protein-1 beta, RANTES, 1309, R83915, R91733, HCCl, T58847, D31065, T64262), CD40 ligand, C-kit Ligand, Collagen, Colony stimulating factor (CSF), Complement factor 5a, Complement inhibitor, Complement receptor 1, cytokines, (e.g., epithelial Neutrophil Activating Peptide-78, GROα/MGSA, GROβ, GROγ, MIP- lα, MIP- 1Δ, MCP-I), Epidermal Growth Factor (EGF), Erythropoietin ("EPO"), Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF), Fibrinogen, Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase, Gonadotropin, growth factors, Hedgehog proteins (e.g., Sonic, Indian, Desert), Hemoglobin, Hepatocyte Growth Factor (HGF), Hirudin, Human serum albumin, Insulin, Insulin-like Growth Factor (IGF), interferons (e.g., IFN-alpha, IFN-beta, IFN-gamma), interleukins (e.g., IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-I, IL-12, etc.), Keratinocyte Growth Factor (KGF), Lactoferrin, leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M, Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones (e.g., Human Growth Hormone), Pleiotropin, Protein A, Protein G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble complement receptor I, Soluble I-CAM 1, Soluble interleukin receptors (IL-I, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15), Soluble TNF receptor, Somatomedin, Somatostatin,

Somatotropin, Streptokinase, Superantigens, i.e., Staphylococcal enterotoxins (SEA, SEB, SECl, SEC2, SEC3, SED, SEE), Superoxide dismutase (SOD), Toxic shock syndrome toxin (TSST-I), Thymosin alpha 1, Tissue plasminogen activator, Tumor necrosis factor beta (TNF-β), Tumor necrosis factor receptor (TNFR), Tumor necrosis factor-alpha (TNF-α), Vascular Endothelial Growth Factor (VEGF), Urokinase and many others.

[0119] One class of proteins that can be made using the compositions and methods for in vivo incorporation of spectroscopically labeled unnatural amino acids described herein includes transcriptional modulators or a portion thereof. Example transcriptional modulators include genes and transcriptional modulator proteins that modulate cell growth, differentiation, regulation, or the like. Transcriptional modulators are found in prokaryotes, viruses, and eukaryotes, including fungi, plants, yeasts, insects, and animals, including mammals, providing a wide range of therapeutic targets. It will be appreciated that expression and transcriptional activators regulate transcription by many mechanisms, e.g., by binding to receptors, stimulating a signal transduction cascade, regulating expression of transcription factors, binding to promoters and enhancers, binding to proteins that bind to promoters and enhancers, unwinding DNA, splicing pre-mRNA, polyadenylating RNA, and degrading RNA. [0120] Another class of proteins of the invention (e.g., proteins with one or more spectroscopically labeled unnatural amino acids) include expression activators such as cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products, e.g., interleukins (e.g., IL-I, IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-alpha, TGF-beta, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4NCAM-1, ICAM- 1/LFA-l, and hyalurin/CD44; signal transduction molecules and corresponding oncogene products, e.g., Mos, Ras, Raf, and Met; and transcriptional activators and suppressors, e.g., p53, Tat, Fos, Myc, Jun, Myb, ReI, and steroid hormone receptors such as those for estrogen, progesterone, testosterone, aldosterone, the LDL receptor ligand and corticosterone. [0121] Enzymes (e.g., industrial enzymes) or portions thereof with at least one spectroscopically labeled unnatural amino acid are also provided by the invention. Examples of enzymes include, but are not limited to, e.g., amidases, amino acid racemases, acylases, dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transferases, haloperoxidases, monooxygenases (e.g., p450s), lipases, lignin peroxidases, nitrile hydratases, nitrilases, proteases, phosphatases, subtilisins, transaminase, and nucleases. [0122] Many of these proteins are commercially available (see, e.g., the Sigma

BioSciences 2004 catalog and price list), and the corresponding protein sequences and genes and, typically, many variants thereof, are well-known (see, e.g., Genbank). Any of them can be modified by the insertion of one or more spectroscopically labeled unnatural amino acid or other unnatural amino acid according to the invention, e.g., to facilitate determination of the protein's structure and/or properties.

[0123] A variety of other proteins can also be modified to include one or more spectroscopically labeled unnatural amino acid. For example, the invention can include substituting one or more natural amino acids in one or more vaccine proteins with a spectroscopically labeled unnatural amino acid, e.g., in proteins from infectious fungi, e.g., Aspergillus, Candida species; bacteria, particularly E. coli, which serves a model for pathogenic bacteria, as well as medically important bacteria such as Staphylococci (e.g., aureus), or Streptococci (e.g., pneumoniae); protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as (+) RNA viruses (examples include Poxviruses e.g., vaccinia; Picomaviruses, e.g. polio; Togaviruses, e.g., rubella; Flaviviruses, e.g., HCV; and Coronaviruses), (-) RNA viruses (e.g., Rhabdo viruses, e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNA viruses (Reoviruses, for example), RNA to DNA viruses, i.e., Retroviruses, e.g., HIV and HTLV, and certain DNA to RNA viruses such as Hepatitis B.

[0124] Agriculturally related proteins such as insect resistance proteins (e.g., the Cry proteins), starch and lipid production enzymes, plant and insect toxins, toxin-resistance proteins, Mycotoxin detoxification proteins, plant growth enzymes (e.g., ribulose 1,5-bisphosphate carboxylase/oxygenase, "RUBISCO"), lipoxygenase (LOX), and phosphoenolpyruvate (PEP) carboxylase are also suitable targets for spectroscopically labeled unnatural amino acid or other unnatural amino acid modification.

[0125] In certain embodiments, the protein of interest (or portion thereof) in the methods and/or compositions of the invention is encoded by a nucleic acid. Typically, the nucleic acid comprises at least one selector codon, at least two selector codons, at least three selector codons, at least four selector codons, at least five selector codons, at least six selector codons, at least seven selector codons, at least eight selector codons, at least nine selector codons, or ten or more selector codons. [0126] Nucleic acids (e.g., genes) coding for proteins of interest can be mutagenized using methods well-known to one of skill in the art and described herein to include, e.g., one or more selector codon for the incorporation of a spectroscopically labeled unnatural amino acid. For example, a nucleic acid for a protein of interest is mutagenized to include one or more selector codon, providing for the insertion of the one or more spectroscopically labeled unnatural amino acids. The invention includes any such variant, e.g., mutant, versions of any protein, e.g., including at least one spectroscopically labeled unnatural amino acid. Similarly, the invention also includes corresponding nucleic acids, i.e., any nucleic acid with one or more selector codon that encodes one or more spectroscopically labeled unnatural amino acid. [0127] To make a protein that includes a spectroscopically labeled unnatural amino acid

(e.g., trifluoromethoxyphenylalanines, etc.), one can use host cells and organisms that are adapted for the in vivo incorporation of the spectroscopically labeled unnatural amino acid via orthogonal tRNA/RS pairs. Host cells are genetically engineered (e.g., transformed, transduced or transfected) with one or more vectors that express the orthogonal tRNA, the orthogonal tRNA synthetase, and a vector that encodes the protein to be derivatized. Each of these components can be on the same vector, or each can be on a separate vector, or two components can be on one vector and the third component on a second vector. The vector can be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide.

V. KITS

[0128] The invention also provides various kits. For example, a kit for producing a protein with a trifluoromethoxyphenylalanine (e.g., p-trifluoromethoxyphenylalanine, etc.) at a specified position is provided, where the kit includes a cell comprising an orthogonal tRNA that functions in the cell and recognizes a selector codon and an orthogonal aminoacyl-tRNA synthetase, packaged in one or more containers. For example, the O-RS may comprise an amino acid sequence of SEQ ID NOS: 17-30 or a conservative variant thereof. In one class of embodiments, the kit further includes a trifluoromethoxyphenylalanine. In another class of embodiments, the kit further comprises instructional materials for producing the protein, an appropriate cell growth medium, reagents for introducing a target nucleic acid encoding the protein of interest and including the selector codon into the cell, or the like. Any composition, system or device of the invention can also be associated with appropriate packaging materials (e.g., containers, etc.) for production in kit form. A kit may also include a plasmid and instructions for practicing a method described herein.

VI. PROTEIN SPECTROSCOPY [0129] As noted above, site-specific, efficient incorporation of spectroscopically labeled unnatural amino acids (e.g., trifluoromethoxyphenylalanines, such as p- trifluoromethoxyphenylalanine, etc.) into proteins facilitates studies of the proteins by spectroscopic techniques, including, but not limited to, NMR spectroscopy, EPR spectroscopy, X-ray spectroscopy, UV spectrometry, mass spectroscopy, fluorescence spectroscopy, and vibrational (e.g., infrared or Raman) spectroscopy.

VII. METHODS USING SPECTROSCOPICALLY LABELED PROTEINS

[0130] Also as noted, one general class of embodiments provides methods for producing a spectroscopically labeled protein, in which methods a nucleic acid that encodes the protein is translated in a translation system. The nucleic acid includes a selector codon. The translation system includes an orthogonal tRNA (O-tRNA) that recognizes the selector codon, an unnatural amino acid comprising a spectroscopic label (e.g., a trifluoromethoxyphenylalanine), and an orthogonal aminoacyl tRNA synthetase (O-RS) (see, e.g., SEQ ID NOS: 17-30) that preferentially aminoacylates the O-tRNA with the unnatural amino acid. The unnatural amino acid is incorporated into the protein as it is translated, thereby producing the spectroscopically labeled protein.

[0131] In this class of embodiments, the methods optionally include subjecting the spectroscopically labeled protein to a spectroscopic technique, including, but not limited to, NMR spectroscopy, EPR spectroscopy, UV spectrometry, X-ray spectroscopy (e.g., for detection of radiation), mass spectroscopy, fluorescence spectroscopy, or vibrational (e.g., infrared or Raman) spectroscopy. As just one example, in one embodiment, the spectroscopically labeled protein comprises a ¹⁵N isotope, and the spectroscopic technique comprises a solvent-exposed amine transverse relaxation optimized spectroscopy (SEA-TROSY) experiment. As another specific example, the spectroscopically labeled protein can comprise a ¹⁹F isotope, and the spectroscopic technique can comprise a one-dimensional non-proton NMR experiment (e.g., to study conformational changes, ligand binding, or the like). Many other spectroscopic techniques (e.g., NMR techniques such as NOESY, HMQC, HSQC, HSQC-NOESY, TROSY, SEA- TROSY, and TROSY-HSQC) are well known in the art and can be adapted for use in the methods of the invention, and many such techniques are described herein.

[0132] In these embodiments, the spectroscopic technique is optionally performed on the spectroscopically labeled protein in vivo, e.g., in intact cells, intact tissue, or the like. Alternatively, the spectroscopic technique can be performed on the spectroscopically labeled protein in vitro, e.g., in a cellular extract, on a purified or partially purified protein, or the like. [0133] In these embodiments, the spectroscopic technique can be used, e.g., to obtain information about the structure, function, abundance, and/or dynamics of the protein, e.g., two- dimensional structure, three-dimensional structure, conformational changes, ligand binding, catalytic mechanism, protein folding, protein concentration, and/or the like. For example, in one class of embodiments, the methods include subjecting the spectroscopically labeled protein to a spectroscopic technique and generating information regarding one or more changes in structure or dynamics of the spectroscopically labeled protein. In some embodiments, the methods include analyzing an interaction between the spectroscopically labeled protein and a ligand or substrate. The interaction can include, e.g., a change in conformation in the spectroscopically labeled protein, binding of a ligand to a specific site near the spectroscopic label, and/or a catalytic reaction performed by the spectroscopically labeled protein.

VIII. METHODS FOR NMR RESONANCE ASSIGNMENT USING ISOTOPICALLY LABELED PROTEINS [0134] Assignment of resonances to particular amino acids in a protein of interest is a key step in NMR studies. Typically, a resonance (an individual signal in an NMR spectrum) is assigned to a particular atom (e.g., the alpha carbon of a particular amino acid) or group of indistinguishable atoms (e.g., the three protons of a methyl group). [0135] Site-specific isotopic labeling of a protein, e.g., using an unnatural amino acid containing an NMR active isotope (e.g., trifluoromethoxyphenylalanines, such as p- trifluoromethoxyphenylalanine, etc.), can greatly simplify the process of resonance assignment, whether many, a few, or even only one resonance is being assigned. For example, in NMR studies of a protein's three-dimensional structure, isotopic labeling of the protein can aid assignment of relevant resonances to their corresponding amino acids, e.g., for resonances difficult to assign by other techniques. As another example, assigning only a single residue (or a small number of residues) at or near an active site, ligand binding site, protein-protein interface, or the like is sometimes desirable, in which case isotopic labeling of the relevant residue(s) can facilitate detailed NMR analysis of even very large proteins. [0136] Accordingly, one general class of embodiments provides methods for assigning NMR resonances to one or more amino acid residues in a protein of interest. In the methods, an unnatural amino acid comprising an NMR active isotope is provided and incorporated, producing an isotopically-labeled protein of interest, in a translation system. The translation system includes a nucleic acid encoding the protein of interest and comprising at least one selector codon for incorporating the unnatural amino acid (e.g., trifluoromethoxyphenylalanines, such as p- trifluoromethoxyphenylalanine, etc.) at a specific site in the protein (e.g., at a selected position in the amino acid sequence of the protein), an orthogonal tRNA (O-tRNA) that recognizes the selector codon, and an orthogonal aminoacyl tRNA synthetase (O-RS) described herein that preferentially aminoacylates the O-tRNA with the unnatural amino acid. An NMR experiment is performed on the isotopically labeled protein, and data generated due to an interaction between the NMR active isotope of the unnatural amino acid and a proximal atom is analyzed, whereby one or more NMR resonances are assigned to one or more amino acid residues in the protein. [0137] Exemplary translation systems including 0-tRNA/O-RS pairs, exemplary selector codons, and exemplary unnatural amino acids have been described above. The NMR active isotope on the unnatural amino acid can be essentially any suitable isotope, including, e.g. ²H, ¹³C, ¹⁵N, ³H, ⁷Li, ¹³B, ¹⁴N, ¹⁷O, ¹⁹F, ²³Na, ²⁷Al, ²⁹Si, ³¹P, ³⁵Cl, ³⁷Cl, ³⁹K, ⁵⁹Co, ⁷⁷Se, ⁸¹Br, ¹¹³Cd, ¹¹⁹Sn, and ¹⁹⁵Pt.

[0138] A variety of NMR techniques are well known in the art and can be applied to the methods of the present invention. For example, the NMR experiment can be an HSQC experiment, a TROSY experiment, a SEA-TROSY experiment, a TROSY-HSQC experiment, a NOESY experiment, an HSQC-NOESY experiment, or any of the other suitable experiments known in the art and/or described herein. [0139] The specific site at which the isotopically labeled unnatural amino acid is incorporated can be essentially any site which is of interest. For example, the specific site of the unnatural amino acid can comprise an active site or ligand binding site of the protein, or it can comprise a site proximal to an active site or ligand binding site of the protein. [0140] The NMR experiment can be performed in vivo or in vitro. Thus, for example, data can be collected in vivo on the isotopically labeled protein, on a cellular extract comprising the isotopically labeled protein, or on a purified or substantially purified isotopically labeled protein.

[0141] A related general class of embodiments also provides methods for resonance assignment. In these methods for assigning an NMR resonance to an amino acid residue occupying a specific position in a protein of interest, the methods include providing a first sample comprising the protein. In this first sample, the protein comprises, at the specific position, an amino acid residue comprising an NMR active isotope. An NMR experiment is performed on the first sample and a first set of data is collected. A second sample comprising the protein is also provided, in which the protein comprises, at the specific position, an unnatural amino acid lacking the NMR active isotope. An NMR experiment is performed on the second sample and a second set of data is collected. The first and second sets of data are compared, whereby a resonance present in the first set and not present in the second set is assigned to the amino acid residue at the specific position.

[0142] In one class of embodiments, the second sample is provided by translating a nucleic acid that encodes the protein in a translation system. The nucleic acid comprises a selector codon for incorporating the unnatural amino acid at the specific position in the protein. The translation system includes an orthogonal tRNA (O-tRNA) that recognizes the selector codon, the unnatural amino acid lacking the NMR active label, and an orthogonal aminoacyl tRNA synthetase (O-RS) that preferentially aminoacylates the O-tRNA with the unnatural amino acid. The NMR active isotope can be, e.g., ¹H, ¹⁵N, ¹³C, or ¹⁹F.

[0143] These methods can be useful for, e.g., resolving ambiguities in resonance assignments, e.g., during determination of the three-dimensional structure of the protein. For example, if resonances are being assigned for a fully ¹⁵N and/or ¹³C labeled protein, the unlabeled unnatural amino acid can be incorporated into an otherwise fully labeled protein, and by the disappearance of the signal from that residue, a resonance can be assigned. For example, the ¹⁵N signal of a particular tyrosine residue could be assigned if that tyrosine is replaced by O- methyl-tyrosine not labeled with ¹⁵N, assuming that incorporation of the unnatural amino acid does not perturb the protein's structure. The methods can also be applied to ¹H spectra, partially ¹⁵N and/or ¹³C labeled proteins, and/or the like.

[0144] Essentially all of the features noted above apply to this embodiment as well, as relevant, e.g., for NMR active isotopes, composition of the translation system, NMR techniques, and the like. As for the embodiments above, the specific position at which the unnatural amino acid is incorporated can be essentially any site which is of interest in the protein.

IX. SPECTROSCOPIC TECHNIQUES

[0145] A variety of spectroscopic techniques are known in the art and can be adapted to the methods of the present invention. Protein NMR spectroscopy, for example, is described in, e.g., Cavanagh et al. (1995) Protein NMR Spectroscopy: Principles and Practice, Academic Press; Levitt (2001) Spin Dynamics: Basics of Nuclear Magnetic Resonance, John Wiley & Sons; Evans (1995) Biomolecular NMR Spectroscopy, Oxford University Press; Wuthrich (1986) NMR of Proteins and Nucleic Acids (Baker Lecture Series), Kurt Wiley-Interscience; Neuhaus and Williamson (2000) The Nuclear Overhauser Effect in Structural and Conformational Analysis, 2nd Edition, Wiley- VCH; Macomber (1998) A Complete Introduction to Modern NMR Spectroscopy, Wiley-Interscience; Downing (2004) Protein NMR Techniques (Methods in Molecular Biology), 2nd edition, Humana Press; Clore and Gronenbom (1994) NMR of Proteins (Topics in Molecular and Structural Biology), CRC Press; Reid (1997) Protein NMR Techniques, Humana Press; Krishna and Berliner (2003) Protein NMR for the Millenium (Biological Magnetic Resonance), Kluwer Academic Publishers; Kiihne and De Groot (2001) Perspectives on Solid State NMR in Biology (Focus on Structural Biology, 1), Kluwer Academic Publishers; and Jones et al. (1993) Spectroscopic Methods and Analyses: NMR, Mass Spectrometry, and Related Techniques (Methods in Molecular Biology, Vol. 17), Humana Press. [0146] A variety of single-dimensional (ID) and multi-dimensional (e.g., 2D, 3D and

4D) NMR spectroscopic techniques have been described, including both solution and solid-state NMR techniques. Such techniques include, e.g., ID heteronuclear correlation experiments, ID heteronuclear filtered experiments, COSY, NOESY, HMQC (heteronuclear multiple quantum correlation spectroscopy), HSQC (heteronuclear single quantum correlation spectroscopy), HMBC (heteronuclear multiple bond correlation spectroscopy), HSQC-NOESY, HETCOR, TROSY (transverse relaxation optimized spectroscopy), SEA-TROSY (solvent-exposed amine transverse relaxation optimized spectroscopy), TROSY-HSQC, CRINEPT-TROSY, CRIPT- TROSY, PISEMA (polarization inversion with spin exchange at the magic angle), MAS (magic angle spinning), and MAOSS (magic angle oriented single spinning), among many others. See, e.g., the above NMR references as well as Wider (2000) BioTechniques 29:1278-1294; Pellecchia et al. (2002) Nature Rev. Drug Discov. (2002) 1:211-219; Arora and Tamm (2001) Curr. Opin. Struct. Biol. 11:540-547; Flaux et al. (2002) Nature 418:207-211; Pellecchia et al. (2001) J. Am. Chem. Soc. 123:4633-4634; and Pervushin et al. (1997) Proc. Natl. Acad. Sci. USA 94:12366-12371.

[0147] A variety of spin-labels have been described in the art, as have a number of uses for spin-labels, e.g., in NMR studies of protein structure and dynamics. For example, NMR resonances of a uniformly isotopically labeled protein that includes a spin-label will be broadened by paramagnetic relaxation enhancement dependent on the distance of the reporter group relative to the spin-label. For a protein of known structure, this method can be used for resonance assignments, especially in conjunction with amino-acid-type selectively labeled protein (similar to the technique described in Cutting et al. (2004) "NMR resonance assignment of selectively labeled proteins by the use of paramagnetic ligands" J. Biomol. NMR 30:205-10). Site-directed introduction of a spin-label into a protein as described herein can also be used to screen for ligand binding to a site near the spin-label (see e.g., the SLAPSTIC method, Jahnke et al. (2001) J. Am. Chem. Soc. 123:3149-50). In addition, paramagnetic relaxation enhancement by site-directed spin-labeling as described herein can provide distance restraints (e.g., long-range distance restraints) for protein structure calculations (Battiste and Wagner (2000) Biochemistry 39:5355-65). This technique can facilitate structure determination by NMR, including structure determination of large proteins, including membrane proteins. It will be evident that the unnatural amino acid comprising the spin-labeled group (whether the group is attached before or after incorporation of the amino acid into the protein) is not typically spectroscopically studied itself; it is the effect of the spin-label on other NMR active nuclei throughout the protein that is typically observed spectroscopically. Introduction of spin-labels site-specifically into proteins using unnatural amino acids, either directly via unnatural amino acids comprising spin-labels or indirectly via unnatural amino acids providing an attachment point for spin-labels, has significant advantages over current methods for introduction of spin-labels (e.g., via S-S bond formation to cysteine mutants); for example, with the methods of the invention, spin-labels can be readily incorporated at sites not occupied (or occupiable) by cysteine residues. Since spin-labels are paramagnetic in their oxidized form but lose their usefulness upon reduction, the labels are typically protected from oxidation, e.g., by attaching the spin-label to the protein in the final step before the NMR measurement of paramagnetic relaxation enhancement. A reference spectrum is typically collected on the reduced form, e.g., after addition of a reducing agent such as ascorbic acid to the NMR sample containing the spin-labeled protein.

[0148] For additional details of spin-labels and NMR, see, e.g., Jahnke (2002) "Spin labels as a tool to identify and characterize protein-ligand interactions by NMR spectroscopy" ChemBioChem 3:167-173; R. A. Dwek (1973) Monographs on Physical Biochemistry: Nuclear Magnetic Resonance (N.M.R.) in Biochemistry. Applications to enzyme systems Oxford

University Press, New York; P. A. Kosen (1989) Methods Enzymol. 177:86; Hubbell (1996) "Watching proteins move using site-directed spin labeling" Structure 4:781; Hustedt and Beth (1999) "Nitroxide spin-spin interactions: Applications to Protein Structure and Dynamics" Annu.l Rev. Biophys. Biomol. Struct. 28:129-153; Berliner, ed. (1976) Spin Labeling: Theory and Applications New York: Academic; Berliner, ed. (1979) Spin Labeling II: Theory and

Applications New York: Academic; Berliner and Reuben, eds. (1989) Biological Magnetic Resonance. Vol. VIII: Spin Labeling Theory and Applications New York: Plenum, including, e.g., Hideg and Hankovszky "Chemistry of spin-labeled amino acids and peptides. Some new mono- and bifunctionalized nitroxide free radicals" pp. 427-488; Hanson et al. (1998) "Electron spin resonance and structural analysis of water soluble, alanine-rich peptides incorporating TOAC" MoI. Phys. 95:95766; Hanson P et al. (1996) "Distinguishing helix conformations in alanine-rich peptides using the unnatural amino acid TOAC and electron spin resonance" J. Am. Chem. Soc. 118:271; Hanson et al. (1996) "ESR characterization of hexameric, helical peptides using double TOAC spin labeling" J. Am. Chem. Soc. 118:7618; Rassat and Rey (1967) Bull. Soc. CHm. France 3:815-817; Jahnke et al. (2001) J. Am. Chem. Soc. 123:3149-3150;

Mchaourab et al. (1996) "Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics" Biochemistry 35:7692-7704; and Columbus et al. (2001) "Molecular motion of spin labeled side chains in alpha-helices: Analysis by variation of side chain structure" Biochemistry 40:3828-3846.

[0149] Chelators for paramagnetic metals and their uses in NMR studies have been similarly well described. They can be used, for example, for NMR protein structure refinement (Donaldson et al. (2001) "Structural characterization of proteins with an attached ATCUN motif by paramagnetic relaxation enhancement NMR spectroscopy" J. Am. Chem. Soc. 123:9843-9847 and Pintacuda et al. (2004) "Site- specific labelling with a metal chelator for protein- structure refinement" J. Biomol. NMR 29:351-361), for resonance assignments (Pintacuda et al. (2004) "Fast structure -based assignment of ¹⁵N HSQC spectra of selectively ¹⁵15N-labeled paramagnetic proteins" J. Am. Chem. Soc. 126:2963-2970), and for magnetically aligning proteins for the measurement of residual dipolar couplings (Barbieri et al. (2002) "Structure-independent cross- validation between residual dipolar couplings originating from internal and external orienting media" J. Biomol. NMR 22:365-368 and Barbieri et al. (2002) "Paramagnetically induced residual dipolar couplings for solution structure determination of lanthanide binding proteins" J. Am. Chem. Soc. 124:5581-5587, and references therein). A reference spectrum is optionally collected on a form of the protein that includes the chelator but not the paramagnetic metal, e.g., before addition of the paramagnetic metal to the chelator.

[0150] EPR spectroscopy (electron paramagnetic resonance spectroscopy, sometimes called electron spin resonance or ESR spectroscopy) is similar to NMR, the fundamental difference being that EPR is concerned with the magnetically induced splitting of electronic spin states, while NMR describes transitions between nuclear spin states. EPR spectroscopy is similarly well described in the literature, as are UV spectrometry, X-ray spectroscopy, mass spectroscopy, fluorescence spectroscopy, and vibrational (e.g., infrared or Raman) spectroscopy. See, e.g., Weil et al. (1994) Electron Paramagnetic Resonance: Elementary Theory and Practical Applications, Wiley-Interscience; Carmona, et al. (1997) Spectroscopy of Biological Molecules: Modern Trends, Kluwer Academic Publishers; Hester et al. (1996) Spectroscopy of Biological Molecules, Special Publication Royal Society of Chemistry (Great Britain); Spiro (1987) Biological Aplications of Raman Spectroscopy, John Wiley & Sons Inc; and Jones et al. (1993) Spectroscopic Methods and Analyses: NMR, Mass Spectrometry, and Related Techniques (Methods in Molecular Biology, Vol. 17), Humana Press.

[0151] A variety of spectrometers are commercially available. For example, NMR spectrometers are available, e.g., from Varian (Palo Alto, Calif.; available on the World Wide Web at varianinc.com) and Bruker (Germany; available on the World Wide Web at bruker.com). X. PROTEIN PURIFICATION

[0152] Spectroscopic analysis of labeled proteins can be performed in vivo or in vitro, on unpurified, partially purified, or purified proteins. When purification of a spectroscopically (e.g., isotopically) labeled protein, or a protein to be so labeled, from the translation system is desired, such purification can be accomplished by any of a number of methods well known in the art, including, e.g., ammonium sulfate or ethanol precipitation, centrifugation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, high performance liquid chromatography (HPLC), gel filtration, hydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography, gel electrophoresis, and the like.

[0153] In addition to other references noted herein, a variety of protein purification methods are well known in the art, including, e.g., those set forth in R. Scopes, Protein Purification, Springer- Verlag, N. Y. (1982); Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc. N. Y. (1990); Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ. ; Harris and Angal (1990) Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3rd Edition Springer Verlag, N. Y.; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition Wiley- VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ. ; and the references cited therein. [0154] Well known techniques for refolding proteins can be used if necessary to obtain the active conformation of the protein when the protein is denatured during intracellular synthesis, isolation or purification. Methods of reducing, denaturing and renaturing proteins are well known to those of skill in the art (see the references above and Debinski, et al. (1993) J. Biol. Chem. 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem. 4:581-585; and Buchner, et al. (1992) Anal. Biochem. 205:263-270).

[0155] The nucleotide sequence encoding the polypeptide can optionally be fused in- frame to a sequence encoding a module (e.g., a domain or tag) that facilitates purification of the polypeptide and/or facilitates association of the fusion polypeptide with a particle, a solid support or another reagent. Such modules include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on and/or binding to immobilized metals (e.g., a hexahistidine tag), a sequence which binds glutathione (e.g., GST), a hemagglutinin (HA) tag (corresponding to an epitope derived from the influenza hemagglutinin protein; see Wilson et al. (1984) Cell 37:767), maltose binding protein sequences, the FLAG epitope utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, Wash.), and the like. The inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and the sequence of the invention is useful to permit removal of the module following, or during, purification of the polypeptide.

XL EXAMPLE

[0156] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Accordingly, the following example is offered to illustrate, but not to limit the claimed invention.

[0157] This example describes methods and compositions of reagents for the incorporation of 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF₃Phe) into proteins using an orthogonal aminoacyl tRNA synthetase/tRNA pair in a position encoded by an TAG amber mutation. In particular, the composition of a novel tRNA synthetase is described for incorporation in E. coli. The utility of the site-specific incorporation OCF₃Phe for fluorine NMR studies of proteins is also illustrated.

MATERIALS AND METHODS [0158] Plasmids and Libraries: All plasmids used in the selection of a OCF₃Phe- specific tRNA synthetase were obtained from the Schultz Lab (see, Wang, L. "Expanding the genetic code of Escherichia coli" Science 2001, 292:498-500, and Xie et al "Adding amino acids to the genetic repertoire" Curr. Opin. Chem.l Biol. 2005, 9:548-554). pREP2- YC- JYCUA encodes the mutated tRNA^Tyr _Cu_A from M. jannaschii, a chloramphenicol resistance gene containing a TAG stop codon, and a tetracycline resistance gene. pNEG encodes the mutated tRNA^TyrcuA, barnase with two TAG stop codons in the coding sequence, and β-lactamase. pBK- MJYRS-Ll, pBK-MJYRS-L2 (Wang, L. "Expanding the genetic code of Escherichia coli" Science 2001, 292:498-500), and pBK-MJYRS-L3D (Schultz et al. "A genetically encoded infrared probe" J. Am. Chem. Soc. 2006, 128:13984-13985) encode the tyrosyl tRNA synthetase (aa RS) from M. jannaschii with different positions of the active site randomized to all 20 amino acids, as previously described (Wang, L. "Expanding the genetic code of Escherichia coli" Science 2001, 292:498-500 and Schultz et al. "A genetically encoded infrared probe" J. Am. Chem. Soc. 2006, 128:13984-13985). pLeiZ encodes the Z-domain protein with a TAG codon at position 7 and C-terminal Hisβ-tag, the M/tRNA^Tyr _Cu_A, and β-lactamase (Wang et al. "Addition of the keto functional group to the genetic code of Escherichia coli" Proc. Natl. Acad. ScL USA 2003, 100:56-61). Unless specified, all chemicals were obtained from Sigma and disposables from Fisher Scientific. OCF₃-DL-PlIe was purchased from JRD Fluorochemicals (Leatherhead, Surrey, U.K.) and used without further purification. [0159] Selection of a novel OCFsPhe-specific tRNA synthetase: In the first round of selection, a frozen aliquot, equivalent to ~10¹⁰ cfu, of DHlOB cells transformed with pREP2-YC- JYCUA and each of the pBK library plasmids were plated on GMML- Agar supplemented with 0.04% glucose; 50 μg/ml kanamycin; 0.25 mM OCF₃Phe (JRD Fluorochemicals); and 50μg/ml chloramphenicol or on LB-Agar with 50 μg/ml kanamycin; 0.5 mM OCF₃Phe; and 50μg/ml chloramphenicol. After 3 days, cells were scraped from the plates and the plasmids purified by Spin Miniprep Kit (Qiagen). The pREP2 and pBK plasmids were separated by gel electrophoresis on a 1% agarose gel, and the pBK plasmid pools were repurified by Minelute Gel Extraction Kit (Qiagen). For negative selections, the pBK pools were transformed by electroporation into HKlOO cells (JCSG; derived from Genehogs by Invitrogen) harboring the pNEG plasmid and plated on LB-Agar supplemented with 50 μg/ml kanamycin, 100 μg/ml ampicillin, and 0.2% arabinose. After 12-14 hours of growth, the pBK plasmid pools were isolated as before and transformed into HKlOO cells with the pREP2 plasmid. After a total of four positive rounds (with 50, 50, 75, and 100 μg/ml chloramphenicol, with 0.25, 0.25, 0.5, and 0.5 mM OCF₃Phe, and with all defined media plates after the first round) and three negative rounds, colonies were picked from the final positive round for further study. A total of 296 colonies were replica plated onto agar plates using the positive selection recipe with 0 mM OCF₃Phe and 20, 35, or 50 μg/ml chloramphenicol; 0.5 mM OCF₃Phe with 100, 125, or 150 μg/ml chloramphenicol; or 1 mM OCF₃Phe with 100, 125, or 150 μg/ml chloramphenicol. The plasmids from 37 of these colonies that showed high growth with unnatural amino acid and poor growth without it were isolated and sequenced, revealing 14 novel sequences (Table 1 and 2). Clones A6 (32V,65A,108Q,109W,158A,162K), B7 (32V,108A,109W,158G,162Q), BlO (32A,65A,108W,109M,158G,159N), F6 (32A,65S,108Q,109A,158A,162Y) and H4 (26I,32V,65G,108H,109Y,158A,162H) were evaluated further based on the combined results of the replica plates and the number of occurrences of the clone. Table 1: DNA sequences of selected OCF₃Phe-specific tRNA synthetases. Nucleotide differences are underlined.

SEQ ID

1 10 20 30 40 50

Al l ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGTTA

61 70 80 90 100 110

1 Library AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTNNNATAGGTTTTGAACCAAGTGGTAAA

2 OCF₃Phe_ _A6 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGTGATAGGTTTTGAACCAAGTGGTAAA

3 OCF₃Phe_ _B6 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGCTATAGGTTTTGAACCAAGTGGTAAA

4 OCF₃Phe_ _B7 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGTGATAGGTTTTGAACCAAGTGGTAAA

5 OCF₃Phe_ _B10 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGCTATAGGTTTTGAACCAAGTGGTAAA

6 OCF₃Phe_ _C2 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGTTATAGGTTTTGAACCAAGTGGTAAA

7 OCF₃Phe_ _D5 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGTTATAGGTTTTGAACCAAGTGGTAAA

8 OCF₃Phe_ _D9 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACCAAGTGGTAAA

9 OCF₃Phe_ _E 7 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGTGATAGGTTTTGAACCAAGTGGTAAA

10 OCF₃Phe_ _F6 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGCTATAGGTTTTGAACCAAGTGGTAAA

11 OCF₃Phe_ _F 7 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTATTATAGGTTTTGAACCAAGTGGTAAA

12 OCF₃Phe_ _F8 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGTGATAGGTTTTGAACCAAGTGGTAAA

13 OCF₃Phe_ _G2 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCATATAGGTTTTGAACCAAGTGGTAAA

14 OCF₃Phe_ _G5 AGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGTGATAGGTTTTGAACCAAGTGGTAAA

15 OCF₃Phe_ _H4 AGAGAGGTTTTAAAAATAGATGAAAAATCTGCTGTGATAGGTTTTGAACCAAGTGGTAAA

121 130 140 150 160 170

Al l ATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTT

181 190 200 210 220 230

1 Library GATATAATTATANNNTTGGCTGATTTANNNGCCTATTTAAACCAGAAAGGAGAGTTGGAT

2 OCF₃Phe_ _A6 GATATAATTATAGCTTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGAT

3 OCF₃Phe_ _B6 GATATAATTATAGCGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGAT

4 OCF₃Phe_ _B7 GATATAATTATACTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGAT

5 OCF₃Phe_ _B10 GATATAATTATAGCTTTGGCTGATTTACATGCCTATTTAAACCAGAAAGGAGAGTTGGAT

6 OCF₃Phe_ _C2 GATATAATTTTAGGTTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGAT

7 OCF₃Phe_ _D5 GATATAATTATACATTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGAT

8 OCF₃Phe_ _D9 GATATAATTATACCGTTGGCTGATTTACATGCCTATTTAAACCAGAAAGGAGAGTTGGAT

9 OCF₃Phe_ _E 7 GATATAATTATATCGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGAT

10 OCF₃Phe_ _F6 GATATAATTATAAGTTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGAT

11 OCF₃Phe_ _F 7 GATATAATTATAACTTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGAT

12 OCF₃Phe_ _F8 GATATAATTATACAGTTGGCTGATTTACATGCCTATTTAAACCAGAAAGGAGAGTTGGAT

13 OCF₃Phe_ _G2 GATATAATTATAGCGTTGGCTGATTTAAATGCCTATTTAAACCAGAAAGGAGAGTTGGAT

14 OCF₃Phe_ _G5 GATATAATTATAACTTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGAT

15 OCF₃Phe_ _H4 GATATAATTATAGGGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGAT

241 250 260 270 280 290

Al l GAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCA

301 310 320 330 340 350

1 Library AAATATGTTTATGGAAGTGAANNNNNNCTTGATAAGGATTATACACTGAATGTCTATAGA

2 OCF₃Phe_ _A6 AAATATGTTTATGGAAGTGAACAGTGGCTTGATAAGGATTATACACTGAATGTCTATAGA

3 OCF₃Phe_ _B6 AAATATGTTTATGGAAGTGAAAAGTGGCTTGATAAGGATTATACACTGAATGTCTATAGA

4 OCF₃Phe_ _B7 AAATATGTTTATGGAAGTGAAGCTTGGCTTGATAAGGATTATACACTGAATGTCTATAGA

5 OCF₃Phe_ _B10 AAATATGTTTATGGAAGTGAATGGATGCTTGATAAGGATTATACACTGAATGTCTATAGA

6 OCF₃Phe_ _C2 AAATATGTTTATGGAAGTGAAGAGTGGCTTGATAAGGATTATACACTGAATGTCTATAGA

7 OCF₃Phe_ _D5 AAATATGTTTATGGAAGTGAAGAGCCTCTTGATAAGGATTATACACTGAATGTCTATAGA

8 OCF₃Phe_ _D9 AAATATGTTTATGGAAGTGAATGGATGCTTGATAAGGATTATACACTGAATGTCTATAGA

9 OCF₃Phe_ _E 7 AAATATGTTTATGGAAGTGAAACGCAGCTTGATAAGGATTATACACTGAATGTCTATAGA

10 OCF₃Phe_ _F6 AAATATGTTTATGGAAGTGAACAGGCGCTTGATAAGGATTATACACTGAATGTCTATAGA

11 OCF₃Phe_ _F 7 AAATATGTTTATGGAAGTGAACGTTGGCTTGATAAGGATTATACACTGAATGTCTATAGA

12 OCF₃Phe_ _F8 AAATATGTTTATGGAAGTGAAAGGGAGCTTGATAAGGATTATACACTGAATGTCTATAGA

13 OCF₃Phe_ _G2 AAATATGTTTATGGAAGTGAATGGATGCTTGATAAGGATTATACACTGAATGTCTATAGA

14 OCF₃Phe_ _G5 AAATATGTTTATGGAAGTGAATTGGGGCTTGATAAGGATTATACACTGAATGTCTATAGA

15 OCF, Phe H4 AAATATGTTTATGGAAGTGAACATTATCTTGATAAGGATTATACACTGAATGTCTATAGA

361 370 380 390 400 410

Al l TTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAG

421 430 440 450 460 470

Library GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGNNNGTTAATNNNNNNCAT OCF₃Phe_A6 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGCGATTCAT 3 OCF₃Phe_ _B6 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTATTCAT

4 OCF₃Phe_ _B7 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGGATTCAT

5 OCF₃Phe_ _B10 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTAATCAT

6 OCF₃Phe_ _C2 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGGATTCAT

7 OCF₃Phe_ _D5 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTCTATTCAT

8 OCF₃Phe_ _D9 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTGCTCAT

9 OCF₃Phe_ _E 7 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGCGATTCAT

10 OCF₃Phe_ _F6 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGCGATTCAT

11 OCF₃Phe_ _F 7 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGCGATTCAT

12 OCF₃Phe_ _F8 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGTCGGTTAATAGTGTGCAT

13 OCF₃Phe_ _G2 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTGCGCAT

14 OCF₃Phe_ _G5 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTCGATTCAT

15 OCF₃Phe_ _H4 GATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGCTATTCAT

481 490 500 510 520 530

1 Library TATNNNGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

2 OCF₃Phe_ _A6 TATAAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

3 OCF₃Phe_ _B6 TATGTTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

4 OCF₃Phe_ _B7 TATCAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

5 OCF₃Phe_ _B10 TATCTTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

6 OCF₃Phe_ _C2 TATGTGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

7 OCF₃Phe_ _D5 TATAGTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

8 OCF₃Phe_ _D9 TATCTTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

9 OCF₃Phe_ _E 7 TATGTGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

10 OCF₃Phe_ _F6 TATTATGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

11 OCF₃Phe_ _F 7 TATTCTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

12 OCF₃Phe_ _F8 TATCATGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

13 OCF₃Phe_ _G2 TATCTTGGGGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

14 OCF₃Phe_ _G5 TATAGTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

15 OCF, Phe H4 TATCATGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCA

541 550 560 570 580 590

All AGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGAT

601 610 620 630 640 650 All GGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAA

661 670 680 690 700 710 All GAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCA

721 730 740 750 760 770 All ATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAA

781 790 800 810 820 830 All TTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAG

841 850 860 870 880 890 All GAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAG

901 910 920 All CCAATTAGAAAGAGATTATAA

Table 2: Protein sequences of selected OCF₃Phe-specific tRNA synthetases. Amino acid differences are underlined.

SEQ ID

1 10 20 30 40 50

16 Library MDEFEMIKRNTSEIISEEELREVLKXDEKSAXIGFEPSGKIHLGHYLQIKKMIDLQNAGF

17 OCF₃PHE- _A6 MDEFEMIKRNTSEIISEEELREVLKKDEKSAVIGFEPSGKIHLGHYLQIKKMIDLQNAGF

18 OCF₃PHE- _B6 MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLGHYLQIKKMIDLQNAGF

19 OCF₃PHE- _B7 MDEFEMIKRNTSEIISEEELREVLKKDEKSAVIGFEPSGKIHLGHYLQIKKMIDLQNAGF

20 OCF₃PHE- _B10 MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLGHYLQIKKMIDLQNAGF

21 OCF₃PHE- _C2 MDEFEMIKRNTSEIISEEELREVLKKDEKSAVIGFEPSGKIHLGHYLQIKKMIDLQNAGF

22 OCF₃PHE- _D5 MDEFEMIKRNTSEIISEEELREVLKKDEKSAVIGFEPSGKIHLGHYLQIKKMIDLQNAGF

23 OCF₃PHE- _D9 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF

24 OCF₃PHE- _E7 MDEFEMIKRNTSEIISEEELREVLKKDEKSAVIGFEPSGKIHLGHYLQIKKMIDLQNAGF

25 OCF₃PHE- _F6 MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLGHYLQIKKMIDLQNAGF

26 OCF₃PHE- _F7 MDEFEMIKRNTSEIISEEELREVLKKDEKSAI-IGFEPSGKIHLGHYLQIKKMIDLQNAGF

27 OCF₃PHE- _F8 MDEFEMIKRNTSEIISEEELREVLKKDEKSAVIGFEPSGKIHLGHYLQIKKMIDLQNAGF

28 OCF₃PHE- _G2 MDEFEMIKRNTSEIISEEELREVLKKDEKSAHIGFEPSGKIHLGHYLQIKKMIDLQNAGF

29 OCF₃PHE- _G5 MDEFEMIKRNTSEIISEEELREVLKKDEKSAVIGFEPSGKIHLGHYLQIKKMIDLQNAGF

30 OCF₃PHE- _H4 MDEFEMIKRNTSEIISEEELREVLKI-DEKSAVIGFEPSGKIHLGHYLQIKKMIDLQNAGF

61 70 80 90 100 110

16 Library DIIXXLADLXAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEXXLDKDYTLNVYR

17 OCF₃PHE- _A6 DIIIALADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEQWLDKDYTLNVYR

18 OCF₃PHE- _B6 DIIIALADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEKWLDKDYTLNVYR

19 OCF₃PHE- _B7 DIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEAWLDKDYTLNVYR

20 OCF₃PHE- _B10 DIIIALADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEWMLDKDYTLNVYR

21 OCF₃PHE- _C2 DIILGLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEEWLDKDYTLNVYR

22 OCF₃PHE- _D5 DIIIHLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEEPLDKDYTLNVYR

23 OCF₃PHE- _D9 DIIIPLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEWMLDKDYTLNVYR

24 OCF₃PHE- _E7 DIIISLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSETQLDKDYTLNVYR

25 OCF₃PHE- _F6 DIIISLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEQALDKDYTLNVYR

26 OCF₃PHE- _F7 DIIITLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSERWLDKDYTLNVYR

27 OCF₃PHE- _F8 DIIIQLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSERELDKDYTLNVYR

28 OCF₃PHE- _G2 DIIIALADLNAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEWMLDKDYTLNVYR

29 OCF₃PHE- _G5 DIIITLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSELGLDKDYTLNVYR

30 OCF₃PHE- _H4 DIIIGLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEHYLDKDYTLNVYR

121 130 140 150 160 170

16 Library LALKTTLKRARRSMELIAREDENPKVAEVIYPIMXVNXXHYXGVDVAVGGMEQRKIHMLA

17 OCF₃PHE- _A6 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNAIHYKGVDVAVGGMEQRKIHMLA

18 OCF₃PHE- _B6 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGIHYVGVDVAVGGMEQRKIHMLA

19 OCF₃PHE- _B7 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGIHYQGVDVAVGGMEQRKIHMLA

20 OCF₃PHE- _B10 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGNHYLGVDVAVGGMEQRKIHMLA

21 OCF₃PHE- _C2 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGIHYVGVDVAVGGMEQRKIHMLA

22 OCF₃PHE- _D5 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNSIHYSGVDVAVGGMEQRKIHMLA

23 OCF₃PHE- _D9 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGAHYLGVDVAVGGMEQRKIHMLA

24 OCF₃PHE- _E7 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNAIHYVGVDVAVGGMEQRKIHMLA

25 OCF₃PHE- _F6 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNAIHYYGVDVAVGGMEQRKIHMLA

26 OCF₃PHE- _F7 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNAIHYSGVDVAVGGMEQRKIHMLA

27 OCF₃PHE- _F8 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMSVNSVHYHGVDVAVGGMEQRKIHMLA

28 OCF₃PHE- _G2 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGAHYLGVDVAVGGMEQRKIHMLA

29 OCF₃PHE- _G5 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNSIHYSGVDVAVGGMEQRKIHMLA

30 OCF₃PHE- _H4 LALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNAIHYHGVDVAVGGMEQRKIHMLA

181 190 200 210 220 230

All RELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGWEGNP

241 250 260 270 280 290 All NFIAVDDSPEEIRAKIKKAYCPAGWEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY

301 310 320 330 Al l EELESLFKNKELHPMDLKNAVAEELIKILEPIRKRLZ

[0160] Test expression of the Z-domain: HKlOO cells co-transformed with pLeiZ and each of the five pBK-OCF₃Phe-RS were grown in 50 ml cultures of TB supplemented with 50 μg/ml kanamycin, 100 μg/ml ampicillin, and with or without 1 mM OCF₃Phe. Cells were harvested after 5 hours induction with 1 rnM IPTG at 3O⁰C. Lysates were prepared in 6M guanidine by sonication and clarified by centrifugation at 20,000 g for 20 minutes. The His- tagged proteins from each culture were purified by Ni-NTA (Qiagen) columns according to the manufacturer's protocol. Denaturant was removed by PD-10 columns (GE Healthcare). The protein samples were next evaluated by SDS-PAGE, Bradford assay (Pierce), and ESI-MS. [0161] Evaluation of misincorporation in Z-domain expression: Protein samples of

Z-domain expressed in the absence or presence of OCF₃PlIe with the five evolved OCF₃Phe-RS were digested with trypsin and subjected to MALDI-MS to evaluate the presence of the N- terminal peptide TSVDNXINK, where X represents the mutated position. The predicted masses for incorporation of OCF₃Phe, Tyr, Phe, and Trp were all monitored in a tandem MS-MS experiment to evaluate misincorporation of the natural aromatic amino acids. The Z-domain protein produced with OCF₃Phe-RS clone A6 in the presence of 1 mM OCF₃Phe was used to verify the sequence of the peptide by collisional MALDI-MS-MS. [0162] Expression of FAS-TE mutants containing OCFsPhe: The coding sequences of a second model protein, FAS-TE, were inserted into pMH4 (JCSG) by the PIPE cloning method (Klock et al., in press). TAG codons were mutated in at eleven specific sites using the same cloning technique. OCF₃Phe-RS clone F6 was inserted into the pDUAL plasmid. For each mutant, the expression plasmid and pDUAL- OCF₃ -Phe plamid were co-transformed into HKlOO cells. Multiple single colonies were picked for growth in 50 ml TB cultures with 100 μg/ml ampicillin and 35 μg/ml chloramphenicol. At ODs₉o=1.0, cultures were moved to 30 ⁰C and supplemented with 1 mM OCF₃Phe from a 0.5 M stock in IN HCl. Thirty minutes later, expression was induced by addition of 0.2% arabinose. After 20 hours, cells were harvested. Lysates were prepared by sonication, clarified by centrifugation, and purified by Ni-NTA under native conditions according to the manufacturer's protocol. Protein samples were evaluated by SDS-PAGE, Bradford assay, and ESI-MS before being exchanged into NMR buffer via PD-10 column and concentrated using Amicon-ultra units (Millipore).

[0163] NMR: FAS-TE samples were prepared at 34 to 168 μM in 20 mM deuterated imidazole (Cambridge Isotope Laboratories) pH 7.0, 100 mM NaCl, 0.5 mM TCEP (Pierce), 10% D₂O (Cambridge Isotope Laboratories). All spectra were recorded on an Avance 400 MHz instrument (Bruker Biospin, Billerica, MA) equipped with a ^/^C/^F/^P-QNP-cryoprobe at 300 K. Spectra were typically recorded with 1024 scans, a recycle delay of 2 s, 8192 complex data points with a sweep width of 20 ppm. Proton decoupling was accomplished using Waltz- 16. RESULTS [0164] Evolution of an OCFsPhe-specific tRNA synthetase: Three libraries of mutants of the M. Jannaschii tyrosyl-tRNA synthetase were subjected to alternating rounds of positive and negative selection in order to isolate clones that specifically recognized OCF₃Phe and not any natural amino acid. Five promising RS clones, A6 (32V,65A,108Q,109W,158A,162K), B7 (32V,108A,109W,158G,162Q), BlO (32A,65A,108W,109M,158G,159N), F6 (32A,65S,108Q,109A,158A,162Y) and H4 (26I,32V,65G,108H,109Y,158A,162H) (Table 1 and 2), were used to express Z-domain protein. In all five cases, the Z-domain was expressed only in the presence of OCF₃Phe, as measured by SDS-PAGE and LC-MS (Figure X). The yield was approximately 6-9 mg/L. The expressed protein was confirmed to contain OCF₃Phe at the expected position by peptide sequencing. Based on MALDI-MS-MS, the ratio of expressed protein containing a misincorporated Tyr, Phe, or Trp compared to the correct OCF₃Phe was greater than 300: 1 for two of the aa-RS clones, A6 and F6 (Figure 3). Clone F6 was subsequently chosen for future use because it consistently produced higher expression yields. [0165] Expression of FAS-TE mutant proteins containing OCFsPhe: In order to express the FAS-TE mutant proteins at high levels, OCF₃Phe-RS clone F6 was cloned into pDUAL plasmids that contained either three (pDUAL3) or six copies (pDUALβ) of the mutant amber tRNA^TyrcuA, respectively (Wang et al. "Expanding the genetic code of Escherichia coli" Science 2001, 292:498-500). In the pDUAL plasmids which were derived from pSUP plasmids (Ryu et al. "Efficient incorporation of unnatural amino acids into proteins in Escherichia coli" Nat. Methods 2006, 3:263-5), over-expression of the specific RS is controlled by an inducible promoter. Eleven FAS-TE mutants were expressed with OCF₃Phe substituted at single sites. Positions Leu2222, Thr2255, Tyr2307, Tyr2343, Tyr2347, Tyr2351, Gln2373, Phe2375, His2408, Thr2450, and Tyr2454 were selected in order to place the unnatural amino acid in solvent exposed positions on all sides of the active site as postulated from a model (unpublished) of the protein in complex with orlistat, a known inhibitor (Kridel "Orlistat is a novel inhibitor of fatty acid synthase with antitumor activity" Cancer Research 2004, 64:2070-5, and Knowles et al. "A fatty acid synthase blockade induces tumor cell-cycle arrest by down-regulating Skp2" J. Biol. Chem. 2004, 279:30540-5). Expression tests of a FAS-TE mutated at position Tyr2454 to OCF₃Phe showed that expression was higher with pDUAL3-OCF₃Phe. Cells transformed with an expression plasmid for each mutant and pDUAL3-OCF₃Phe were fed the unnatural amino acid and induced to express the model proteins (Table 3). Each mutant was purified using Ni- affinity and size-exclusion chromatography. The average yield of purified protein was 2 mg per

50 ml culture. With the exception of Gln2373OCF₃Phe, purified protein from only one 50 ml culture was transferred to NMR buffer resulting in NMR samples of typically 0.1 rnM in concentration (total volume 0.55 ml). The predicted molecular weight of the mutant proteins was confirmed by ESI-MS (Table 3).

Table 3: Expression yields and molecular weights of OCF₃PlIe FAS-TE mutants. Concentrations of the NMR samples used in Figure 4 are also listed.

[0166] ID NMR spectra of OCF₃-Phe mutant proteins: ID ¹⁹F-NMR spectra were recorded for each FAS-TE mutant (Figure 4). In each case, a single peak was observed within 0.89 ppm of a resonance line recorded for a 0.5 mM solution of the unnatural amino acid, OCF₃Phe dissolved in the same buffer and identical conditions. The width of the fluorine resonance line varied for each position presumably because of conformational exchange. The chemical shift of fluorine resonances is highly sensitive to changes in the environment (Gerig et al. "Fluorine NMR of Proteins" Prog.Nucl. Magn. Reson. Spectr. 1994, 26:293-370, Frieden et al. "The preparation of 19F-labeled proteins for NMR studies" Methods Enzymol. 2004, 380:400- 15, and Danielson et al. "Use of F-19 NMR to Probe Protein Structure and Conformational Changes" Annu. Rev. Biophy. Biomol. Struct. 1996, 25:163-195) and the linewidths are a sensitive monitor of conformational exchange and reflect conformational fluctuations in the protein. Resonance lines that are most affected correspond to residues that have high B-factors or missing electron densities in the available crystal structures (2325 to 2330, 2344 to 2356, 2452 to 2457, and 2487 are missing in the published X-ray structure; PDB file IXKT), suggesting that these regions of the protein indeed undergo conformational exchange. Since the natural amino acid is replaced with the unnatural amino acid OCF₃Phe, some of these effects may be the result of this mutation. At many of the positions, OCF₃Phe incorporation simply adds a hydrophobic trifluoromethyl group to the polar, solvent exposed hydroxyl group of tyrosine residues. This constitutes a very conservative mutation but could cause local perturbations of the surface. These results indicate that OCF₃Phe can be incorporated at all eleven sites in a 33 kDa protein with good yields. Using standard shake flask incubation, single NMR samples were obtained from 50 ml of E. coli culture for 10 of the 11 mutants using only 12.5 mg of the unnatural amino acid. Fresh samples can readily be prepared, e.g., for future binding experiments with small molecule binders. The data also suggests that OCF₃Phe ¹⁹F NMR spectra may be used to probe structural and dynamic rearrangements in large proteins.

[0167] The three different NMR-active unnatural amino acids, ¹³C/¹⁵N-labeledp- methoxy-phenylalanine (OMePhe), ¹⁵N-labeled o-nitrobenzyl-tyrosine (oNBTyr) and OCF₃Phe, were used to study the binding of a small molecule ligand to the thioesterase domain of fatty acid synthetase (FAS-TE), a 33 kDa protein of pharmaceutical interest. Fatty acid synthetase (FAS) is a large, multi-domain enzyme essential for the synthesis of long-chain fatty acids. Because the sequence and architecture of FAS differ significantly between bacteria and mammals, bacterial FAS has long been considered a valuable target for the development of novel antibiotics. In humans, FAS is over-expressed in many cancers and is a drug target for obesity and related diseases. Orlistat, an approved obesity drug, exhibits antitumor activity by inhibiting FAS-TE. We compare orlistat's and compound l's interactions with FAS-TE to evaluate the utility of unnatural amino acids for the characterization of protein-ligand interactions. Successful incorporation of three different NMR-active unnatural amino acids at 11 different positions around the proposed binding site in FAS-TE demonstrates the general utility of the approach to studies of protein structure, dynamics, function and binding of small molecules in particular.

[0168] FAS-TE binding to compound 1: Since orlistat is very insoluble in aqueous solutions, our binding experiments were performed with a FAS-TE binder, compound 1. Compound 1 was synthesized according to Scheme I. Briefly, Garner's aldehyde 9 was treated with a Grignard reagent, affording benzyl alcohol 10 quantitatively, followed by the global deprotecion by aqueous HCl. The amino group of 11 was masked as azide by trifluoromethanesulfonyl azide; the configuration of the stereo center of the amino group was retained under the condition.⁴⁴ The primary hydroxyl group of 12 was selectively converted to the nosylate, and then to the cyclohexylamino group by nucleophilic displacement. The resulting intermediate, 14, was elaborated to the final compound, 17, by following the sequence of benzoylation, azide reduction, and ureation. Although we were unable to determine the diastereo selectivity of the first reaction (9 to 10), the H-NMR and LC-MS analysis of 13 indicated that 13 comprizes mainly one diastereomer. Since the synthesis started with homochiral aldehyde 9 and no steps were considered to involve racemization, we assume the final compound, 17, also comprises mainly one species regarding the benzilic and homobenzylic positions.

Scheme III

[0169] Conditions: (a) 3, 4-(methylenedioxy)phenylmagnesium bromide, THF/toluene

(1:1 v/v), -78 to 23°C, 98% ; (b) IM HCl, THF, 70⁰C; (c) trifluoromethanesulfonyl azide, CuSO₄ , K₂CO₃, H₂OMeOH, O to 23°C, 42% (2 steps); (d) 4-nitrobenzene-l-sulfonyl chloride, pyridine, cat. DMAP, DCM, 23°C, 55%; (e) cyclohexanamine, NMP, 80⁰C; (f) 2,4-difluorobenzoyl chloride, DIEA, DCM, 23°C, 65%; (g) H₂, 5% Pd/C, MeOH, 23°C; (h) n-butylisocyanate, DIEA, DCM, 23°C, 28% (2 steps).

[0170] NMR sample preparation and data collection: As a lock solvent, 50 μL of

D₂O were added to 500 μL of protein solution resulting in a final buffer composition of 18 mM d-imidazole, 91 mM NaCl, 0.45 mM TCEP, pH 6.5. Aliquots of compound 1 were added from a 50 mM stock in deuterated DMSO to a final concentration of 1.4 molar equivalents unless mentioned otherwise. All spectra were recorded at 300 K. ¹⁹F spectra were recorded on an Avance 400 MHz instrument (Bruker Biospin, Billerica, MA) equipped with a ¹HZ¹³CZ¹⁹FZ³¹P- QNP-cryoprobe. Spectra were typically recorded with 1024 scans, a recycle delay of 2 s, 8192 complex data points with a sweep width of 20 ppm. Proton decoupling was accomplished using Waltz- 16. All other spectra were recorded on an Avance 600 MHz instrument equipped with a i_H/i3_c/i5_{N Tχi cryoprobe} i_H_i3_{c HS}Q_{C were typically} recorded with 32 scans, 128 t_i experiments at 300 K using a spectral width of 50 ppm in the carbon dimension and 13.97 ppm in the proton dimension. Similarly, ¹H-¹⁵N HSQC spectra were recorded using 48 scans, 256 ti experiments using a spectral width of 32 ppm and 13.97 ppm in the nitrogen and proton dimension respectively. [0171] Effects of unnatural amino acid incorporation on protein structure: NMR spectra of FAS-TE mutants individually labeled at 11 positions with three different unnatural amino acids were recorded in the absence and presence of a FAS-TE binding compound 1. Data supports the conclusion that incorporation of unnatural amino acids at the 11 sites chosen appears not to negatively affect the structural integrity of these FAS-TE mutants.

[0172] NMR spectra of OCF₃-Phe mutant proteins: ID ¹⁹F-NMR spectra were recorded for each OCF₃PlIe FAS-TE mutant. In each case, a single peak was observed within 0.9 ppm of a resonance line recorded for a 0.5 mM solution of the unnatural amino acid, OCF₃Phe dissolved in the same buffer and identical conditions. The width of the fluorine resonance line varied for each position again suggesting conformational exchange. The chemical shift of fluorine resonances is highly sensitive to changes in the environment and the line widths are a sensitive monitors of conformational exchange and reflect conformational fluctuations in the protein. [0173] Addition of compound 1 results in significant chemical shift changes for residues near the binding site. In some cases, for example, for Tyr-2343-OCF₃Phe, Tyr-2351-OCF₃Phe, His-2408-OCF₃Phe and Tyr-2454-OCF₃Phe compound addition results in sharpening of the fluorine line; for Gln-2373-OCF₃Phe and Phe-2375-OCF₃Phe the opposite is observed. As with Tyr-2307-OMePhe, no changes are observed for Tyr-2307-OCF₃Phe suggesting again that the addition of the bulky OCF₃ (or OMe) group to this near active site residue blocks binding. The small chemical shift changes to Thr-2450-OCF₃Phe and Thr-2255-OCF₃Phe may be a solvent effect as compound 1 is added as DMSO stock (<0.5% final DMSO concentration). [0174] Mapping the binding site of Compound 1: Compound 1 binds tightly to all

FAS-TE mutants (except for Tyr-2307-OMePhe and Tyr-2307-OCF₃Phe). Binding occurs in the slow binding regime for all ¹H, ¹³C, ¹⁵N and ¹⁹F resonances. The chemical shift changes induced by compound l's addition map the binding site of the ligand to the active site of FAS-TE. Data obtained from ¹H-¹³C, ¹H-¹⁵N and ¹⁹F NMR spectra are all consistent in that His-2408, Thr-2450 and Thr-2255 mutants are not affected by binding. The most pronounced chemical shift changes occur at residue Tyr-2343 that is apparently disordered in the uncomplexed proteins (by NMR but not in the crystal structure of the orlistat complex). Increasingly smaller chemical shift changes are observed for neighboring Tyr-2347 and Tyr-2351 mutants. Compound 1 binding most likely stabilizes this flexible region of the protein as suggested by sharper fluorine resonance lines at residues Tyr-2343, Tyr-2351 and Tyr-2454. Modest chemical shift changes are also observed for Gln-2373 and Phe-2375 mutants at the top surface of the binding pocket. At the far right site of the pocket, among the largest chemical shift changes are observed for Leu- 2222 mutants while Thr-2255 and His-2408 mutant resonances are not shifted by compound l's binding. On the other end, unnatural amino acids at residue Thr-2450 and Tyr-2454 are also not affected and consistent with the overall features of the orlistat complex. [0175] Chemical shift mapping of a small molecule binding site in FAS-TE:

Although different in absolute value, trends and magnitude of the chemical shift, changes induced by binding compound 1 in OMePhe, OCF₃PlIe and oNBTyr mutants agree and identify the active site of FAS-TE as the binding pocket. Data obtained from ¹H-¹³C, ¹H-¹⁵N and ¹⁹F NMR spectra are all consistent in that residues His-2408, Thr-2450 and Thr-2255 mutants are not affected by binding. When compared to the crystal structure of the FAS-TE complex with orlistat, Thr-2255 and His-2408 are at least 12 A from the closest orlistat atom while the distance to Thr-2450 is 16 A. Similarly, mutants of Thr-2450 exhibits minimal chemical shift changes as this residue is more than 13 A from any orlistat atom. These observations indicate that compound 1 must bind in the active site not unlike orlistat. The similarities between the orlistat complexes and the complex of compound 1 are further supported by large chemical shift changes for Leu-2222 mutants. Leu-2222, therefore likely marks the extent of the compound binding pocket on the right side. The residue with the most pronounced chemical shift changes for its mutants, Tyr-2343 forms close contacts with the hexanoyl tail of orlistat in its hydrolyzed form and with both hexanoyl and the beginning of the palmitic core in the covalent orlistat complex. Tyr-2343, together with Tyr-2347 and Tyr-2351 are part of an alpha-helix that is observed in only one of two asymmetric units of an unpublished crystal structure and is absent in all published structures. Based on the sharper resonance lines, it is possible that compound 1 binding stabilizes this helix but a more detailed analysis must await a co-crystal structure or more detailed NMR studies. [0176] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A composition comprising an orthogonal aminoacyl-tRNA synthetase (O-RS) that preferentially aminoacylates an orthogonal tRNA (O-tRNA) with trifluoromethoxyphenylalanine.

2. The composition of claim 1, wherein the O-RS comprises an amino acid sequence of SEQ ID NO: 16, wherein X₂₆ is K or I; X₃₂ is V, A, L, I, or H; X₆₄ is I or L; X₆₅ is A, G, L, H, P, S, T, or Q; X₇₀ is H or N; X₁₀₈ is Q, K, A, W, E, T, Q, R, L, or H; X₁₀₉ is W, M, P, Q, A, G, or Y; X₁₅₅ is Q or S; X₁₅₈ is A, G, or S; X₁₅₉ is I, N, A, or V; and X₁₆₂ is K, V, Q, L, V, S, Y, or H.

3. The composition of claim 1, wherein the O-RS comprises an amino acid sequence selected from the group consisting of: SEQ ID NOS: 17-30.

4. The composition of claim 1, wherein the O-RS is encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 2-15.

5. The composition of claim 1, comprising a translation system.

6. The composition of claim 1, comprising the trifluoromethoxyphenylalanine.

7. The composition of claim 6, wherein the trifluoromethoxyphenylalanine is 2-amino-3-(4- (trifluoromethoxy)phenyl)propanoic acid (OCF₃Phe).

8. The composition of claim 1, comprising the O-tRNA, which O-tRNA recognizes a selector codon.

9. The composition of claim 8, wherein the selector codon is an amber codon.

10. The composition of claim 1, comprising a cell.

11. The composition of claim 10, wherein the cell is an Escherichia coli (E. coli) cell.

12. A nucleic acid encoding an aminoacyl-tRNA synthetase (O-RS) that preferentially aminoacylates an orthogonal tRNA (O-tRNA) with trifluoromethoxyphenylalanine.

13. The nucleic acid of claim 12, comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 2-15 and a polynucleotide sequence that hybridizes under highly stringent conditions over substantially an entire length of a nucleotide sequence of SEQ ID NOS: 2-15.

14. The O-RS encoded by the nucleic acid of claim 12.

15. The O-RS of claim 14, comprising an amino acid sequence of SEQ ID NO: 16, wherein X₂₆ is K or I; X₃₂ is V, A, L, I, or H; X₆₄ is I or L; X₆₅ is A, G, L, H, P, S, T, or Q; X₇₀ is H or N; X₁₀₈ is Q, K, A, W, E, T, Q, R, L, or H; X_i09 is W, M, P, Q, A, G, or Y; Xj₅₅ is Q or S; Xi₅₈ is A,

G, or S; Xi₅₉ is I, N, A, or V; and Xi₆₂ is K, V, Q, L, V, S, Y, or H.

16. The O-RS of claim 14, comprising an amino acid sequence selected from the group consisting of: SEQ ID NOS: 17-30.

17. The O-RS of claim 14, comprising an improved K_m and/or K_cat for the trifluoromethoxyphenylalanine relative to a natural amino acid.

18. A method of producing a spectroscopically labeled protein, the method comprising translating a nucleic acid that encodes a protein in a translation system, which nucleic acid comprises a selector codon and which translation system comprises an orthogonal tRNA (O- tRNA) that recognizes the selector codon, trifluoromethoxyphenylalanine, and an orthogonal aminoacyl-tRNA synthetase (O-RS) that preferentially aminoacylates the O-tRNA with the trifluoromethoxyphenylalanine, thereby producing the spectroscopically labeled protein.

19. The method of claim 18, wherein the trifluoromethoxyphenylalanine is 2-amino-3-(4- (trifluoromethoxy)phenyl)propanoic acid (OCF₃PlIe).

20. The method of claim 18, wherein the O-RS comprises an amino acid sequence selected from the group consisting of: SEQ ID NOS: 17-30.

21. The method of claim 18, further comprising analyzing the spectroscopically labeled protein.

22. The method of claim 21, comprising subjecting the spectroscopically labeled protein to a spectroscopic technique.

23. The method of claim 22, wherein the spectroscopic technique comprises a nuclear magnetic resonance (NMR) technique.

24. A method of producing an orthogonal aminoacyl-tRNA synthetase (O-RS), the method comprising:

(a) generating a library of variant aminoacyl-tRNA synthetase (RS) molecules derived from at least one RS; and, (b) selecting or screening the library of variant RS molecules to identify one or more members that aminoacylate an orthogonal tRNA (O-tRNA) with trifluoromethoxyphenylalanine 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF₃Phe), thereby producing the O-RS.

25. The method of claim 24, wherein the trifluoromethoxyphenylalanine is 2-amino-3-(4- (trifluoromethoxy)phenyl)propanoic acid (OCF₃Phe).

26. A compound selected from N-(3-(benzo[d][l,3]dioxol-5-yl)-2-(3-butylureido)-3- hydroxypropyl)-N-cyclohexyl-2,4-difluorobenzamide and the pharmaceutically acceptable salts thereof.