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Incorporation of non-natural modules into proteins: structural features beyond the genetic code

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An Erratum to this article was published on 04 July 2009

Abstract

The biotechnological application of enzymes necessitates a permanent quest for new biocatalysts. Among others, improvement of catalytic activity, modification of substrate specificity, or increase in stability of the enzymes are desirable goals. The exploration of homologous enzymes from various sources or DNA-based methods, like site-directed mutagenesis or directed evolution, yield an incredible variety of biocatalysts but they all rely on the restricted number of canonical amino acids. Chemistry offers an almost unlimited palette of additional modifications which can endow the proteins with improved or even completely new properties. Numerous techniques to furnish proteins with non-natural amino acids or non-proteinogenic modules have been introduced and are reviewed with special focus on expressed protein ligation, a method that combines the potential of protein biosynthesis and chemical synthesis.

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Abbreviations

dmP:

5,5′-Dimethylproline

EPL:

Expressed protein ligation

IPL:

Intein-mediated protein ligation

MESNA:

2-Mercaptoethanesulfonic acid

NCL:

Native chemical ligation

SPPS:

Solid phase peptide synthesis

References

  • An SSA, Cathy CC, Peng J-L, Li Y-J, Rothwarf DM, Welker E, Thannhauser TW, Zhang LS, Tam JP, Scheraga HA (1999) Retention of the cis proline conformation in tripeptide fragments of bovine pancreatic ribonuclease A containing a non-natural proline analogue, 5, 5-dimethylproline. J Am Chem Soc 121:11558–11566

    Article  CAS  Google Scholar 

  • Anthony-Cahill SJ, Magliery TJ (2002) Expanding the natural repertoire of protein structure and function. Curr Pharm Biotechnol 3:299–315

    Article  PubMed  CAS  Google Scholar 

  • Arnold U, Hinderaker MP, Nilsson BL, Huck BR, Gellman SH, Raines RT (2002a) Protein prosthesis: a semisynthetic enzyme with a beta-peptide reverse turn. J Am Chem Soc 124:8522–8523

    Article  PubMed  CAS  Google Scholar 

  • Arnold U, Hinderaker MP, Raines RT (2002b) Semisynthesis of ribonuclease A using intein-mediated protein ligation. Sci World J 2:1823–1827

    CAS  Google Scholar 

  • Arnold U, Hinderaker MP, Köditz J, Golbik R, Ulbrich-Hofmann R, Raines RT (2003) Protein prosthesis: a nonnatural residue accelerates folding and increases stability. J Am Chem Soc 125:7500–7501

    Article  PubMed  CAS  Google Scholar 

  • Arnold U, Köditz J, Markert Y, Ulbrich-Hofmann R (2005) Local fluctuations vs. global unfolding of proteins investigated by limited proteolysis. Biocatal Biotransform 23:159–167

    Article  CAS  Google Scholar 

  • Boerema DJ, Tereshko VA, Kent SB (2008) Total synthesis by modern chemical ligation methods and high resolution (1.1 Å) X-ray structure of ribonuclease A. Biopolymers 90:278–286

    Article  PubMed  CAS  Google Scholar 

  • Brooks B, Phillips RS, Benisek WF (1998) High-efficiency incorporation in vivo of tyrosine analogues with altered hydroxyl acidity in place of the catalytic tyrosine-14 of Δ5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: effects of the modifications on isomerase kinetics. Biochemistry 37:9738–9742

    Article  PubMed  CAS  Google Scholar 

  • Budisa N, Minks C, Medrano FJ, Lutz J, Huber R, Moroder L (1998) Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: structure and stability of the per-thiaproline mutant of annexin V. Proc Natl Acad Sci USA 95:455–459

    Article  PubMed  CAS  Google Scholar 

  • Chung YJ, Huck BR, Christianson LA, Stanger HE, Krauthäuser S, Powell DR, Gellman SH (2000) Stereochemical control of hairpin formation in β-peptides containing dinipecotic acid reverse turn segments. J Am Chem Soc 122:3995–4004

    Article  CAS  Google Scholar 

  • Cotton GJ, Muir TW (2000) Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation. Chem Biol 7:253–261

    Article  PubMed  CAS  Google Scholar 

  • Dawson PE, Muir TW, Clark-Lewis I, Kent SB (1994) Synthesis of proteins by native chemical ligation. Science 266:776–779

    Article  PubMed  CAS  Google Scholar 

  • Dirksen A, Dawson PE (2008) Expanding the scope of chemoselective peptide ligations in chemical biology. Curr Opin Chem Biol 12:760–766

    Article  PubMed  CAS  Google Scholar 

  • Eijsink VG, Bjork A, Gaseidnes S, Sirevag R, Synstad B, van den Burg B, Vriend G (2004) Rational engineering of enzyme stability. J Biotechnol 113:105–120

    Article  PubMed  CAS  Google Scholar 

  • Espinosa JF, Gellman SH (2000) A designed β-hairpin containing a natural hydrophobic cluster. Angew Chem Int Ed Engl 39:2330–2333

    Article  PubMed  CAS  Google Scholar 

  • Evans TC Jr, Xu MQ (2002) Mechanistic and kinetic considerations of protein splicing. Chem Rev 102:4869–4883

    Article  CAS  Google Scholar 

  • Evans TC Jr, Benner J, Xu MQ (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci 7:2256–2264

    Article  PubMed  CAS  Google Scholar 

  • Evans TC Jr, Martin D, Kolly R, Panne D, Sun L, Ghosh I, Chen L, Benner J, Liu XQ, Xu MQ (2000) Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803. J Biol Chem 275:9091–9094

    Article  PubMed  CAS  Google Scholar 

  • Fetrow JS (1995) Omega loops: nonregular secondary structures significant in protein function and stability. FASEB J 9:708–717

    PubMed  CAS  Google Scholar 

  • Flavell RR, Muir TW (2009) Expressed protein ligation (EPL) in the study of signal transduction, ion conduction, and chromatin biology. Acc Chem Res 42:107–116

    Article  PubMed  CAS  Google Scholar 

  • Gauthier MA, Klok HA (2008) Peptide/protein–polymer conjugates: synthetic strategies and design concepts. Chem Commun (Camb) 23:2591–2611

    Article  Google Scholar 

  • Gieselman MD, Xie L, van Der Donk WA (2001) Synthesis of a selenocysteine-containing peptide by native chemical ligation. Org Lett 3:1331–1334

    Article  PubMed  CAS  Google Scholar 

  • Golbik R, Yu C, Weyher-Stingl E, Huber R, Moroder L, Budisa N, Schiene-Fischer C (2005) Peptidyl prolyl cis/trans-isomerases: comparative reactivities of cyclophilins, FK506-binding proteins, and parvulins with fluorinated oligopeptide and protein substrates. Biochemistry 44:16026–16034

    Article  PubMed  CAS  Google Scholar 

  • Gutte B, Merrifield RB (1971) The synthesis of ribonuclease A. J Biol Chem 246:1922–1941

    PubMed  CAS  Google Scholar 

  • Hackenberger CP, Schwarzer D (2008) Chemoselective ligation and modification strategies for peptides and proteins. Angew Chem Int Ed Engl 47:10030–10074

    Article  PubMed  CAS  Google Scholar 

  • Hackeng TM, Griffin JH, Dawson PE (1999) Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology. Proc Natl Acad Sci USA 96:10068–10073

    Article  PubMed  CAS  Google Scholar 

  • Hahn U, Palm GJ, Hinrichs W (2004) Old codons, new amino acids. Angew Chem Int Ed Engl 43:1190–1193

    Article  PubMed  CAS  Google Scholar 

  • Hirata R, Ohsumk Y, Nakano A, Kawasaki H, Suzuki K, Anraku Y (1990) Molecular structure of a gene, VMA1, encoding the catalytic subunit of H+-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem 265:6726–6733

    PubMed  CAS  Google Scholar 

  • Hondal RJ (2005) Incorporation of selenocysteine into proteins using peptide ligation. Protein Pept Lett 12:757–764

    Article  PubMed  CAS  Google Scholar 

  • Kalia J, Abbott NL, Raines RT (2007) General method for site-specific protein immobilization by Staudinger ligation. Bioconjug Chem 18:1064–1069

    Article  PubMed  CAS  Google Scholar 

  • Kane PM, Yamashiro CT, Wolczyk DF, Neff N, Goebl M, Stevens TH (1990) Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase. Science 250:651–657

    Article  PubMed  CAS  Google Scholar 

  • Kent SB (2009) Total chemical synthesis of proteins. Chem Soc Rev 38:338–351

    Article  PubMed  CAS  Google Scholar 

  • Klabunde T, Sharma S, Telenti A, Jacobs WR Jr, Sacchettini JC (1998) Crystal structure of GyrA intein from Mycobacterium xenopi reveals structural basis of protein splicing. Nat Struct Biol 5:31–36

    Article  PubMed  CAS  Google Scholar 

  • Köhn M, Breinbauer R (2004) The Staudinger ligation—a gift to chemical biology. Angew Chem Int Ed Engl 43:3106–3116

    Article  PubMed  Google Scholar 

  • Koide H, Yokoyama S, Kawai G, Ha JM, Oka T, Kawai S, Miyake T, Fuwa T, Miyazawa T (1988) Biosynthesis of a protein containing a nonprotein amino acid by Escherichia coli: l-2-aminohexanoic acid at position 21 in human epidermal growth factor. Proc Natl Acad Sci USA 85:6237–6241

    Article  PubMed  CAS  Google Scholar 

  • Liu W, Brock A, Chen S, Schultz PG (2007) Genetic incorporation of unnatural amino acids into proteins in mammalian cells. Nat Methods 4:239–244

    Article  PubMed  CAS  Google Scholar 

  • Liu D, Xu R, Dutta K, Cowburn D (2008) N-terminal cysteinyl proteins can be prepared using thrombin cleavage. FEBS Lett 582:1163–1167

    Article  PubMed  CAS  Google Scholar 

  • Lue RY, Chen GY, Zhu Q, Lesaicherre ML, Yao SQ (2004) Site-specific immobilization of biotinylated proteins for protein microarray analysis. Methods Mol Biol 264:85–100

    PubMed  CAS  Google Scholar 

  • Marshall GR, Feng JA, Kuster DJ (2008) Back to the future: ribonuclease A. Biopolymers 90:259–277

    Article  PubMed  CAS  Google Scholar 

  • Merrifield BR (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc 85:2149–2154

    Article  CAS  Google Scholar 

  • Milton RC, Milton SC, Kent SB (1992) Total chemical synthesis of a d-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity. Science 256:1445–1448

    Article  PubMed  CAS  Google Scholar 

  • Muir TW (2003) Semisynthesis of proteins by expressed protein ligation. Annu Rev Biochem 72:249–289

    Article  PubMed  CAS  Google Scholar 

  • Muir TW, Sondhi D, Cole PA (1998) Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci USA 95:6705–6710

    Article  PubMed  CAS  Google Scholar 

  • Müller G, Gurrath M, Kurz M, Kessler H (1993) βVI turns in peptides and proteins: a model peptide mimicry. Proteins 15:235–251

    Article  PubMed  Google Scholar 

  • Nilsson BL, Kiessling LL, Raines RT (2000) Staudinger ligation: a peptide from a thioester and azide. Org Lett 2:1939–1941

    Article  PubMed  CAS  Google Scholar 

  • Nilsson BL, Hondal RJ, Soellner MB, Raines RT (2003) Protein assembly by orthogonal chemical ligation methods. J Am Chem Soc 125:5268–5269

    Article  PubMed  CAS  Google Scholar 

  • Nilsson BL, Soellner MB, Raines RT (2005) Chemical synthesis of proteins. Annu Rev Biophys Biomol Struct 34:91–118

    Article  PubMed  CAS  Google Scholar 

  • Pal M, Dasgupta S (2003) The nature of the turn in omega loops of proteins. Proteins 51:591–606

    Article  PubMed  CAS  Google Scholar 

  • Perler FB (1998) Protein splicing of inteins and hedgehog autoproteolysis: structure, function, and evolution. Cell 92:1–4

    Article  PubMed  CAS  Google Scholar 

  • Perler FB (2002) InBase: the intein database. Nucleic Acids Res 30:383–384

    Article  PubMed  CAS  Google Scholar 

  • Perler FB (2005) Protein splicing mechanisms and applications. IUBMB Life 57:469–476

    Article  PubMed  CAS  Google Scholar 

  • Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, Neff N, Noren CJ, Thorner J, Belfort M (1994) Protein splicing elements: inteins and exteins—a definition of terms and recommended nomenclature. Nucleic Acids Res 22:1125–1127

    Article  PubMed  CAS  Google Scholar 

  • Pokorski JK, Jenkins LM, Feng H, Durell SR, Bai Y, Appella DH (2007) Introduction of a triazole amino acid into a peptoid oligomer induces turn formation in aqueous solution. Org Lett 9:2381–2383

    Article  PubMed  CAS  Google Scholar 

  • Quaderer R, Sewing A, Hilvert D (2001) Selenocysteine-mediated native chemical ligation. Helv Chim Acta 84:1197–1206

    Article  CAS  Google Scholar 

  • Ralle M, Berry SM, Nilges MJ, Gieselman MD, van der Donk WA, Lu Y, Blackburn NJ (2004) The selenocysteine-substituted blue copper center: spectroscopic investigations of Cys112SeCys Pseudomonas aeruginosa azurin. J Am Chem Soc 126:7244–7256

    Article  PubMed  CAS  Google Scholar 

  • Saxon E, Armstrong JI, Bertozzi CR (2000) A “traceless” Staudinger ligation for the chemoselective synthesis of amide bonds. Org Lett 2:2141–2143

    Article  PubMed  CAS  Google Scholar 

  • Skrisovska L, Allain FH (2008) Improved segmental isotope labeling methods for the NMR study of multidomain or large proteins: application to the RRMs of Npl3p and hnRNP L. J Mol Biol 375:151–164

    Article  PubMed  CAS  Google Scholar 

  • Soellner MB, Nilsson BL, Raines RT (2006) Reaction mechanism and kinetics of the traceless Staudinger ligation. J Am Chem Soc 128:8820–8828

    Article  PubMed  CAS  Google Scholar 

  • Srinivasan G, James CM, Krzycki JA (2002) Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 296:1459–1462

    Article  PubMed  CAS  Google Scholar 

  • Staudinger H, Meyer J (1919) Über neue organische Phosphorverbindungen III. Phosphinmetylenderivate und Phosphinimine. Helv Chim Acta 2:635–646

    Article  CAS  Google Scholar 

  • Tam JP, Yu Q (1998) Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones. Biopolymers 46:319–327

    Article  PubMed  CAS  Google Scholar 

  • Tam A, Arnold U, Soellner MB, Raines RT (2007a) Protein prosthesis: 1,5-disubstituted[1,2,3]triazoles as cis-peptide bond surrogates. J Am Chem Soc 129:12670–12671

    Article  PubMed  CAS  Google Scholar 

  • Tam A, Soellner MB, Raines RT (2007b) Water-soluble phosphinothiols for traceless Staudinger ligation and integration with expressed protein ligation. J Am Chem Soc 129:11421–11430

    Article  PubMed  CAS  Google Scholar 

  • Valiyaveetil FI, MacKinnon R, Muir TW (2002) Semisynthesis and folding of the potassium channel KcsA. J Am Chem Soc 124:9113–9120

    Article  PubMed  CAS  Google Scholar 

  • Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43

    Article  PubMed  CAS  Google Scholar 

  • Wang L, Xie J, Schultz PG (2006) Expanding the genetic code. Annu Rev Biophys Biomol Struct 35:225–249

    Article  PubMed  Google Scholar 

  • Wu H, Hu Z, Liu XQ (1998) Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc Natl Acad Sci USA 95:9226–9231

    Article  PubMed  CAS  Google Scholar 

  • Xie J, Schultz PG (2005) Adding amino acids to the genetic repertoire. Curr Opin Chem Biol 9:548–554

    Article  PubMed  CAS  Google Scholar 

  • Zawadzke LE, Berg JM (1992) A racemic protein. J Am Chem Soc 114:4002–4003

    Article  CAS  Google Scholar 

  • Ziaco B, Pensato S, D’Andrea LD, Benedetti E, Romanelli A (2008) Semisynthesis of dimeric proteins by expressed protein ligation. Org Lett 10:1955–1958

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The author would like to thank Ronald T. Raines, UW-Madison, and Renate Ulbrich-Hofmann, Martin-Luther University Halle, for their steady cooperation, and Gary Case, UW Biotechnology Center, for the help in peptide synthesis. The German Academic Exchange Service is acknowledged for financial support (D/0104622).

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  1. Institute of Biochemistry and Biotechnology, Martin-Luther University, Kurt-Mothes-Str. 3, 06120, Halle, Germany

    Ulrich Arnold

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  1. Ulrich Arnold
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Correspondence to Ulrich Arnold.

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An erratum to this article can be found at http://dx.doi.org/10.1007/s10529-009-0069-3

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Arnold, U. Incorporation of non-natural modules into proteins: structural features beyond the genetic code. Biotechnol Lett 31, 1129–1139 (2009). https://doi.org/10.1007/s10529-009-0002-9

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