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Synthesis Of Proteins Containing Unnatural Amino Acids Michigan State University Department of Chemistry Justas Jancauskas Why Introduce Unnatural Amino Acids? • Drug industry: N-methylated proteins are stable to proteolysis • Selective protein labeling with antigenic or fluorophoric tags • Selective protein posttranslational modification • Probing protein structure and function relationship van Maarseveen, J. H.; Back, J. W. Angew. Chem. Int. Ed. 2003, 42, 5926–5928. Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.; King D. S.; Horn, D. M.; Schultz, P. G. J. Am. Chem. Soc. 2003, 125, 935–939. How Fast Is Polypeptide Biosynthesis? Electron micrograph • Polymerization rate : 18 aa/sec • 300 amino acid polypeptide in 20 sec in E. coli; • 100 amino acid polypeptide in 1 min. in mammals. • Protein synthesis represents ~30 % of ATP utilized by E. coli. http://arethusa.unh.edu/bchm752/ppthtml/april27/april27/sld025.htm. Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990. Outline ¸ Protein Biosynthesis ¸ Unnatural Amino Acid Incorporation ¸ Chemical Acylation ¸ Exploiting Cell Machinery ¸ Using Existing Genetic Code ¸ First Unnatural Organism ¸ Future Directions Outline ¸ Protein Biosynthesis ¸ Unnatural Amino Acid Incorporation ¸ Chemical Acylation ¸ Exploiting Cell Machinery ¸ Using Existing Genetic Code ¸ First Unnatural Organism ¸ Future Directions The Central Dogma of Molecular Biology Replication DNA messenger RNA (mRNA) transfer RNA (tRNA) ribosomal RNA (rRNA) Transcription RNA Protein Translation Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990. 20 natural L-amino acids O 5' -O P O O3' Base O OH H DNA vs. RNA DNA RNA O 5' -O P O O3' Base O 2' OH OH NH2 N NH2 N N H Adenine N N H N N Guanine Guanine N NH2 NH2 N N Cytosine N H O O Cytosine O O NH N H N N H NH2 NH2 N H NH O NH N H Adenine N O N N O NH Thymine N H O Uracil Main Elements For Protein Biosynthesis • mRNA: template. • Ribosome: polymerase. • tRNACGAAla : amino acid-charged tRNA: • tRNA •Amino acid •RS: aminoacyl-tRNA synthetase, specific for each amino acid and capable of charging most of tRNAs specific for the same amino acid. Lewin, B. Genes VII; Oxford: New York, Oxford University Press, 2000. Transfer RNA (tRNA) Acceptor or amino acid stem. A 7 bp stem that includes 5’-terminal nucleotide. All tRNAs terminate with the sequence CCA with a free 3’-OH group. Anticodon arm. A 5 bp stem ending in the loop that contains the anticodon. Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990. Codon–Anticodon Interaction tRNAUACVal anticodon 3' mRNA 5' C A U G U A • The proper tRNA is selected only through codon–anticodon interactions 5' 3' Charged Transfer RNA ( aa-tRNA) H O R C C _ NH3 O + + ATP H O O R C C O P O Ribose Adenine + PPi _ NH3 O + • Charging of tRNA is performed by aminoacyl-tRNA synthetase (aa-RS). •Many of aa-tRNA synthetases have proofreading function Aminoacyl-adenylate (Aminoacyl-AMP) Isoleucyl-tRNA synthetase: 1 Val / 50,000 Ile tRNA NH2 N tRNA O O P _O O N O 3' 2' O OH O C H C R NH3 + Aminoacyl-tRNA N N CH3 O H3C O NH3 + Isoleucine _ CH3 O H3C O NH3 + Valine Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990. _ Mechanism of Polypeptide Synthesis OH tRNA(n-1) Exit site NH Rn-1 CH O C NH Rn CH O C NH Rn-1 CH O C NH Rn CH O C O NH2 Rn+1 CH O C O tRNA(n) Peptidyl-tRNA site tRNA(n+1) Acceptor site OH tRNA(n) Exit site NH Rn+1 CH O C O NH2 Rn+2 CH O C O tRNA(n+1) tRNA(n+2) Peptidyl-tRNA site Acceptor site Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990. Outline ¸ Protein Biosynthesis ¸ Unnatural Amino Acid Incorporation ¸ Chemical Acylation ¸ Exploiting Cell Machinery ¸ Using Existing Genetic Code ¸ First Unnatural Organism ¸ Future Directions Chemical Acylation of tRNA 5' (amino acid)-AC–C UAC ATG 5' 3' 5' (amino acid)-AC-PO3H HO–C 5' UAC ATG 3' Heckler, T. G.; Chang, L. -H.; Zama, Y.; Naka, T.; Chorghade, M. S.; Hecht. S. M. Biochemistry, 1984, 23, 1468–1473. Chemical Acylation of tRNA R (NVOC)HN H3CO R ClCH2CN COOH NEt3 CN O NO2 O H3CO DMF Et3N Cl O 6-nitroveratrylcarbonyl chloride (NVOC-Cl) N O N O N N O _ O O P O N N O N OH OH O _ O O P _O O O 5'-phospho-2-deoxycytidylyl(3',5')adenosine pdCpA O _ O P O _ O N N N N N pdCpA H2N H2N H2N H2N O (NVOC)HN _ O P O O O O R NH(NVOC) O O OH Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722–2729. Chemical Acylation of tRNA H2N H2N N O N N N N O O _ O P O _ O 5' N O _ O P O O O R NH(NVOC) O O OH HO–C T7 5' 3' tRNA gene T7 RNA polymerase T4 RNA ligase UAC 5' (aa)-AC–C UAC Bain, J. D.; Wacker, D. A.; Kuo, E. E.; Lyttle, M. H.; Chamberlin, A. R. J. Org. Chem, 1991, 56, 4615–4625. Cload, S. T.; Liu, D. R.; Froland, W. A.; Schultz, P. G. Chem. Biol. 1996, 3, 1033–1038. Unnatural Amino Acid Incorporation 5' (aa)-ACC 5' UAG 3' In vitro translation Protein containing unnatural amino acid mRNA 5' • Possible AUC UAG 3' multiple incorporation of one unnatural amino acid • mRNA is unstable • Need stoichiometric amounts of aminoacylated-tRNA. Bain, J. D.; Wacker, D. A.; Kuo, E. E.; Lyttle, M. H.; Chamberlin, A. R. J. Org. Chem, 1991, 56, 4615–4625. Cload, S. T.; Liu, D. R.; Froland, W. A.; Schultz, P. G. Chem. Biol. 1996, 3, 1033–1038. Outline ¸ Protein Biosynthesis ¸ Unnatural Amino Acid Incorporation ¸ Chemical Acylation ¸ Exploiting Cell Machinery ¸ Using Existing Genetic Code ¸ First Unnatural Organism ¸ Future directions Finding Unique Codon Three STOP codons do not encode an amino acid. UAA – ochre UAG – amber UGA – opal Lewin, B. Genes VII; Oxford: New York, Oxford University Press, 2000. Amber Codon Suppression 5' (aa)-ACC In vitro / in vivo translation 5' AUC UAG Protein containing unnatural amino acid 3' Requirements for amber suppression : • tRNACUA and RS must be orthogonal toE. coli system • The resulting tRNACUAaa must recognize amber codon at the same rate as in normal protein synthesis Selecting For Orthogonal tRNACUA TAG ApR S. cerevisiae Gln-RNA synthetase Grown in the presence of ampicillin sc-tRNACUAGln survivors encode a suppressor tRNA capable of inserting Gln in response to the TAG codon IC50 500 mg mL-1 E. coli TAG ApR Grown in the presence of ampicillin no growth observed sc-tRNACUAGln IC50 20 mg mL-1 E. coli Liu, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4780–4785. Verifying the Orthogonality of GlnRS S. cerevisiae Gln-RNA synthetase Genomic GlnRS deletion no growth observed E. coli E. coli Gln-RNA synthetase Genomic GlnRS deletion cells growth E. coli Liu, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4780–4785. Other tRNACUA/RS Orthogonal Pairs Table 1. IC50 values (mg/mL) for control experiments E. coli without suppressor tRNACUA E. coli with supF expression 9.7 1700 Table 2. IC50 values (mg/mL) of screening experiments tRNA source tRNACUA Saccharomyces cerevisae tRNATyr 234 Homo sapiens tRNATyr 1206 tRNACUA/aaRS Saccharomyces cerevisiae tRNAGln 20 500 Methanococcus jannaschii tRNATyr 12 400 Liu, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4780–4785. Wang, L.; Magliery, T. J.; Liu, D. R.; Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 5010–5011 Wang, L.; Schultz, P. G. Chem. Biol. 2001, 8, 883–890. Engineering a Synthetase With Unnatural Amino Acid Specificity Engineering the specificity of synthetase Rational design Combinatorial approach Difficult due to high fidelity of natural synthetase Generate synthetase mutant library Select on their specificity for unnatural amino acid Engineering a Synthetase With Unnatural Amino Acid Specificity TAG M. jannaschii Tyr-RS mutant library ApR orthogonal mj-tRNACUATyr add unnatural amino acid positive selection/screen survivors encode synthetases that charge the orthogonal suppressor tRNA with natural or unnatural amino acid E. coli Wang, L.; Schultz, P. G. Chem. Biol. 2001, 8, 883–890. Engineering a Synthetase With Unnatural Amino Acid Specificity survivors encode synthetases that charge the orthogonal suppressor tRNA with natural or unnatural amino acid TAG barnase survivors encode synthetase that charge the orthogonal suppressor tRNA with unnatural amino acid only No unnatural Amino acid added negative selection/screening synthetase from positive selection E. coli Wang, L.; Schultz, P. G. Chem. Biol. 2001, 8, 883–890. orthogonal mj-tRNACUATyr Engineering a Synthetase With Unnatural Amino Acid Specificity TAG M. jannaschii Tyr-RS mutant library ApR orthogonal mj-tRNACUATyr add unnatural amino acid positive selection/screen survivors encode synthetases that charge the orthogonal suppressor tRNA with natural or unnatural amino acid E. coli mutagenesis, DNA shuffling survivors encode synthetase that charge the orthogonal suppressor tRNA with unnatural amino acid only TAG barnase No unnatural Amino acid added negative selection/screening synthetase from positive selection E. coli Wang, L.; Schultz, P. G. Chem. Biol. 2001, 8, 883–890. orthogonal mj-tRNACUATyr Outline ¸ Protein Biosynthesis ¸ Unnatural Amino Acid Incorporation ¸ Chemical Acylation ¸ Exploiting Cell Machinery ¸ Using Existing Genetic Code ¸ First Unnatural Organism ¸ Future directions One Step Towards First “Unnatural” Organism TAG dihydrofolate reductase mj-Tyr-RNA synthetase In vivo translation SDS-PAGE gel and Western blot analysis mj-tRNATyr CUA E. coli O OH H3CO NH2 mj-tRNACUATyr + + + – + mutTyrRS – + + + – O-met-L-Tyr – + – + + wtTyrRS + – – – – O-methyl-L-tyrosine Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Science, 2001, 292, 498–500. Synthesis of p-aminophenylalanine (p-AF) O HO HO OH O OH O OH OH O OH O O OH OH OH O OH O PapA OH O O NH2 NH2 O Glucose 4-amino-4-deoxychorismate chorismic acid O PapB 4-amino-4deoxyprephenic acid PapC NH2 O OH Proteins Papa, PapB and PapC convert chorismate to paminophenylpyruvate O OH aminotransferase O NH 2 p-aminophenylalanine NH2 p-aminophenylpyruvate Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.; King D. S.; Horn, D. M.; Schultz, P. G. J. Am. Chem. Soc. 2003, 125, 935–939. The First “Unnatural” Organism sperm whale myoglobin TAG Plpp mjTyr-RNA synthetase papA papB papC In vivo translation SDS-PAGE gel and Western blot analysis mjtRNATyr CUA E. coli Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.; King D. S.; Horn, D. M.; Schultz, P. G. J. Am. Chem. Soc. 2003, 125, 935–939. The First “Unnatural” Organism wt-tRNATyr + – – – – – – TyrRS + – – – – – – pAFRS – – + – + + – mutRNACUATyr – + + + + + + pAF – – + + – – – Plpp papA,papB,papC – – – – – + + 2 3 4 5 6 7 8 Protein yield Wild-type: 3.5 mg/L Mutant: 3.0 mg/L 1 For the first time bacterium has: • the ability to synthesize pAF from simple carbon sources; • an aa-tRNA synthetase that uniquely utilizes pAF and no other amino acid; • a tRNA that is acylated by this synthetase and no other; • the same tRNA delivers pAF efficiently into proteins in response to the amber codon, TAG Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.; King D. S.; Horn, D. M.; Schultz, P. G. J. Am. Chem. Soc. 2003, 125, 935–939. Outline ¸ Protein Biosynthesis ¸ Unnatural Amino Acid Incorporation ¸ Chemical Acylation ¸ Exploiting Cell Machinery ¸ Using Existing Genetic Code ¸ First Unnatural Organism ¸ Future Directions Wobble Property of tRNA “Wobble” is the property that allows one anticodon to pair with two or three codons tRNAVal anticodon 3' mRNA C A U G U A 5' 5' 3' or mRNA 5' G U G 3' Table 3. Allowed wobble pairing combinations in the third codon–anticodon position 5’-anticodon base 3’-codon base C G A U U A or G G U or C I U, C or A Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990. The Genetic Code Lewin, B. Genes VII; Oxford: New York, Oxford University Press, 2000. Using Wobble Property L-proline is encoded by UUC and UUU One tRNA with GAA anticodon recognizes both codons Generation of tRNA with AAA anticodon might recognize UUU codon with higher efficiency Murine dihydrofolate reductase (mDHFR) gene contains four UUC and five UUU codons mDHFR had five substituted proline residues with L-3-(2-naphthyl)alanine O H2N OH L-3-(2-naphthyl)alanine Kwon, I.; Kirshenbaum, K.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 7512–7513 The Genetic Code 3 nt code gives 64 possible codons 4 nt code would give 256 codons 5 nt code would give 1024 codons Lewin, B. Genes VII; Oxford: New York, Oxford University Press, 2000. 4 and 5 nt Codons –CGC UAU GUA CUU ACC GGC CGU UAU GAC– Arg Tyr Val Leu Thr Gly Arg Tyr Asp Mutation at Thr position –CGC UAU GUA CUU AGGU GGC CGU UAU GAC– Arg Tyr Val Leu Xaa Gly Arg Tyr Asp Hohsaka, T.; Ashizuka, Y.; Sasaki, H.; Murakami, H.; Sisido, M. J. Am. Chem. Soc. 1999, 121, 12194–12195. Magliery, T. J.; Anderson, J. C.; Schultz, P. G. J. Mol. Biol. 2001, 307, 755–769. Increasing the Codon Size (4 nt codon) tRNA Codons selected CUUCCUAA AGGA CGCUAGGA CUAG AUUCUAAC UAGA CUGCCUAU AGGC GUAGGGUA CCCU IC50 500 mg mL-1 Suppression: 2.5–35 % Magliery, T. J.; Anderson, J. C.; Schultz, P. G. J. Mol. Biol. 2001, 307, 755–769. Increasing the Codon Size (5 nt codon) Anticodon loop Codon Suppression (%) CUGUCCUAA AGGAC 5.0 CUGUCCUAA AGGAU 11.3 CUAUUGGAC CCAAU 4.4 UUGGUGGAA CCACC 1.6 CUAGUGGAC CCACU 7.4 GUGAUCCAA CCAUC 8.0 UUGAUGGAG CCAUC 5.6 CUGAGGGUC CCCUC 3.8 UUGACCGAC CGGUC 4.5 GUGGUAGGA CUACC 7.4 UUAGUAGAU CUACU 11.2 CUGAUAGAA CUAGC 8.5 UUACUAGAC CUAGU 12.0 Anderson, J. C.; Magliery, T. J.; Schultz P. G. Chem. Biol. 2002, 9, 237–244. Novel Base Pairs Requirements for the third base pair: • stable and selective base pairing • unnatural nucleoside must be membrane permeable • must be stable inside the cell • efficient and high fidelity enzymatic incorporation into DNA N N 7-aza-indole Tae, E. L.; Wu, Y.; Xia G.; Schultz, P.G.; Romesberg, F. E. J. Am. Chem. Soc. 2001, 123, 7439–7440. Summary and Conclusions Chemical acylation: • mRNA is not stable • Need stoichiometric amount of charged tRNA Exploiting cell machinery: • orthogonal tRNA/RS pair is required • engineered pair is capable to suppress amber codon by inserting unnatural amino acid Protein engineering: • evolving better catalysts • synthesizing proteins bearing D-amino acids • synthesis of glycoproteins OH HO HO O O NHAc NH2 OH O b-N-acetylglucosamine (GlcNAc)-L-serine Zhang, Z.; Gildersleeve, J.; Yang, Y.; Xu, R.; Loo, J. A.; Uryu, S.; Wong, C., Schultz, P. G. Science 2004, 303, 371–373. Acknowledgments Prof. John Frost Dr. Karen Frost Frost Group Members Dr. Jihane Achkar Wei Niu Jiantao Guo Ningqing Ran Xiaofei Jia Heather Stueben Man Kit Lau Dr. Dongming Xie Kin Sing Stephen Lee Jinsong Yang Wensheng Li Dr. Jian Yi Mapitso Molefe