Expressed protein ligation: a resourceful tool to study protein structure and function



This review outlines the use of expressed protein ligation (EPL) to study protein structure, function and stability. EPL is a chemoselective ligation method that allows the selective ligation of unprotected polypeptides from synthetic and recombinant origin for the production of semi-synthetic protein samples of well-defined and homogeneous chemical composition. This method has been extensively used for the site-specific introduction of biophysical probes, unnatural amino acids, and increasingly complex post-translational modifications. Since it was introduced 10 years ago, EPL applications have grown increasingly more sophisticated in order to address even more complex biological questions. In this review, we highlight how this powerful technology combined with standard biochemical analysis techniques has been used to improve our ability to understand protein structure and function.


Chemical ligation Protein α-thioesters Intein Protein splicing Circular polypeptides Post-translational modifications Isotopic labeling 


  1. 1.
    Hackenberger CP, Schwarzer D (2008) Chemoselective ligation and modification strategies for peptides and proteins. Angew Chem Int Ed Engl 47:10030–10074PubMedGoogle Scholar
  2. 2.
    Camarero JA (2006) New developments for the site-specific attachment of proteins to surfaces. Biophys Rev Lett 1:1–28Google Scholar
  3. 3.
    Dawson PE, Muir TW, Clark-Lewis I, Kent SBH (1994) Synthesis of proteins by native chemical ligation. Science 266:776–779PubMedGoogle Scholar
  4. 4.
    Tam JP, Lu YA, Liu CF, Shao J (1995) Peptide synthesis using unprotected peptides through orthogonal coupling methods. Proc Natl Acad Sci USA 92:12485–12489PubMedGoogle Scholar
  5. 5.
    Wieland T, Bokelmann E, Bauer L, Lang HU, Lau H (1953) Polypeptide synthesis. VIII. Formation of sulfur containing peptides by intramolecular migration of aminoacyl groups. Liebigs Ann Chem 583:129–149Google Scholar
  6. 6.
    Wieland T (1988) Sulfur in biomimetic peptide synthesis. In: Kleinkauf Jaeniche VD (eds) The roots of modern biochemistry. Walter de Gruyter, Berlin, pp 213–221Google Scholar
  7. 7.
    Kent SB (2009) Total chemical synthesis of proteins. Chem Soc Rev 38:338–351PubMedGoogle Scholar
  8. 8.
    Erlanson DA, Chytil M, Verdine GL (1996) The leucine zipper domain controls the orientation of AP-1 in the NFAT•AP-1•DNA complex. Chem Biol 3:981–991PubMedGoogle Scholar
  9. 9.
    Muir TW, Sondhi D, Cole PA (1998) Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci USA 95:6705–6710PubMedGoogle Scholar
  10. 10.
    Evans TC, Benner J, Xu M-Q (1999) The in Vitro ligation of bacterially expressed proteins using an intein from Metanobacterium thermoautotrophicum. J Biol Chem 274:3923–3926PubMedGoogle Scholar
  11. 11.
    Flavell RR, Muir TW (2009) Expressed protein ligation (EPL) in the study of signal transduction, ion conduction, and chromatin biology. Acc Chem Res 42:107–116PubMedGoogle Scholar
  12. 12.
    Pellois JP, Muir TW (2006) Semisynthetic proteins in mechanistic studies: using chemistry to go where nature can’t. Curr Opin Chem Biol 10:487–491PubMedGoogle Scholar
  13. 13.
    Muralidharan V, Muir TW (2006) Protein ligation: an enabling technology for the biophysical analysis of proteins. Nat Methods 3:429–438PubMedGoogle Scholar
  14. 14.
    Tsukiji S, Nagamune T (2009) Sortase-mediated ligation: a gift from Gram-positive bacteria to protein engineering. Chembiochem 10:787–798PubMedGoogle Scholar
  15. 15.
    Saleh L, Perler FB (2006) Protein splicing in cis and in trans. Chem Rec 6:183–193PubMedGoogle Scholar
  16. 16.
    Evans TC Jr, Xu MQ (1999) Intein-mediated protein ligation: harnessing nature’s escape artists. Biopolymers 51:333–342PubMedGoogle Scholar
  17. 17.
    Camarero JA, Muir TW (1999) Native chemical ligation of polypeptides. Curr Protoc Protein Sci 15:18.4.1–18.4.21Google Scholar
  18. 18.
    Dawson PE, Kent SB (2000) Synthesis of native proteins by chemical ligation. Annu Rev Biochem 69:923–960PubMedGoogle Scholar
  19. 19.
    Muir TW (2003) Semisynthesis of proteins by expressed protein ligation. Annu Rev Biochem 72:249–289PubMedGoogle Scholar
  20. 20.
    Hackeng TM, Griffin JH, Dawson PE (1999) Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology. Proc Natl Acad Sci USA 96:10068–10073PubMedGoogle Scholar
  21. 21.
    Hojo H, Aimoto S (1991) Polypeptide Synthesis Using the S-Alkyl Thioester of a Partially Protected Peptide Segment. Synthesis of the DNA-Binding Domain of c-Myb Protein (142–193)-NH2. Bull Chem Soc Jpn 64:111–117Google Scholar
  22. 22.
    Camarero JA, Cotton GJ, Adeva A, Muir TW (1998) Chemical ligation of unprotected peptides directly form a solid support. J Pept Res 51:303–316PubMedGoogle Scholar
  23. 23.
    Camarero JA, Adeva A, Muir TW (2000) 3-Thiopropionic acid as a highly versatile multidetachable thioester resin linker. Int J Pept Res Ther 7:17–21Google Scholar
  24. 24.
    Huse M, Holford MN, Kuriyan J, Muir TW (2000) Semisynthesis of hyperphosphorylated type I TGF beta receptor: addressing the mechanism of kinase activation. J Am Chem Soc 122:8337–8338Google Scholar
  25. 25.
    Shin Y, Winans KA, Backes BJ, Kent SBH, Ellman JA, Bertozzi CR (1999) Fmoc-based synthesis of Peptide-aThioesters: application to the total chemical synthesis of a glycoprotein by native chemical ligation. J Am Chem Soc 121:11684–11689Google Scholar
  26. 26.
    Tolbert TJ, Wong C-H (2000) Intein-mediated synthesis of proteins containing carbohydrates and other molecular probes. J Am Chem Soc 122:5421–5428Google Scholar
  27. 27.
    Miller JS, Dudkin VY, Lyon GJ, Muir TW, Danishefsky SJ (2003) Toward fully synthetic N-linked glycoproteins. Angew Chem Int Ed 42:431–434Google Scholar
  28. 28.
    Camarero JA, Mitchell AR (2005) Synthesis of proteins by native chemical ligation using Fmoc-based chemistry. Protein Pept Lett 12:723–728PubMedGoogle Scholar
  29. 29.
    Ingenito R, Bianchi E, Fattori D, Pessi A (1999) Solid phase synthesis of peptide C-terminal thioesters by Fmoc/t-Bu chemistry Source. J Am Chem Soc 121:11369–11374Google Scholar
  30. 30.
    Camarero JA, Hackel BJ, de Yoreo JJ, Mitchell AR (2004) Fmoc-based synthesis of peptide alpha-thioesters using an aryl hydrazine support. J Org Chem 69:4145–4151PubMedGoogle Scholar
  31. 31.
    Blanco-Canosa JB, Dawson PE (2008) An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation. Angew Chem Int Ed Engl 47:6851–6855PubMedGoogle Scholar
  32. 32.
    Kawakami T, Aimoto S (2009) Peptide ligation via the in situ transformation of an amide into a thioester at a cysteine residue. Adv Exp Med Biol 611:117–118PubMedGoogle Scholar
  33. 33.
    Gesquièe J-C, Diesis E, Tartar A (1990) Conversion of N-terminal cysteine to thiazolidine carboxylic acid during hydrogen fluoride deparotection of peptides containing N-bom protected histidine. J Chem Soc Chem Commun 20:1402–1403Google Scholar
  34. 34.
    Pentelute BL, Gates ZP, Dashnau JL, Vanderkooi JM, Kent SB (2008) Mirror image forms of snow flea antifreeze protein prepared by total chemical synthesis have identical antifreeze activities. J Am Chem Soc 130:9702–9707PubMedGoogle Scholar
  35. 35.
    Severinov K, Muir TW (1998) Expressed protein ligation, a novel method for studying protein–protein interactions in transcription. J Biol Chem 273:16205–16209PubMedGoogle Scholar
  36. 36.
    Evans TC, Benner J, Xu M-Q (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci 7:2256–2264PubMedGoogle Scholar
  37. 37.
    Xu M-Q, Perler FB (1996) The mechanism of protein splicing and its modulation by mutation. EMBO J 15:5146–5153PubMedGoogle Scholar
  38. 38.
    Chong S, Mersha FB, Comb DG, Scott ME, Landry D, Vence LM, Perler FB, Benner J, Kucera RB, Hirvonen CA, Pelletier JJ, Paulus H, Xu MQ (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192:271–281PubMedGoogle Scholar
  39. 39.
    Chong S, Montello GE, Zhang A, Cantor EJ, Liao W, Xu M-Q, Benner J (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucleic Acid Res 26:5109–5115PubMedGoogle Scholar
  40. 40.
    Valiyaveetil FI, MacKinnon R, Muir TW (2002) Semisynthesis and folding of the potassium channel KcsA. J Am Chem Soc 124:9113–9120PubMedGoogle Scholar
  41. 41.
    Camarero JA, Shektman A, Campbell E, Chlenov M, Gruber TM, Bryant DA, Darst SA, Cowburn D, Muir TW (2002) Autoregulation of a bacterial sigma factor explored using segmental isotopic labeling and NMR. Proc Natl Acad Sci USA 99:8536–8541PubMedGoogle Scholar
  42. 42.
    Hirel PH, Schmitter MJ, Dessen P, Fayat G, Blanquet S (1989) Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc Natl Acad Sci USA 86:8247–8251PubMedGoogle Scholar
  43. 43.
    Dwyer MA, Lu W, Dwyer JJ, Kossiakoff AA (2000) Biosynthetic phage display: a novel protein engineering tool combining chemical and genetic diversity. Chem Biol 7:263–274PubMedGoogle Scholar
  44. 44.
    Iwai H, Pluckthun A (1999) Circular b-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett 459:166–172Google Scholar
  45. 45.
    Camarero JA, Fushman D, Cowburn D, Muir TW (2001) Peptide chemical ligation inside living cells: in vivo generation of a circular protein domain. Bioorg Med Chem 9:2479–2484PubMedGoogle Scholar
  46. 46.
    Cotton GJ, Ayers B, Xu R, Muir TW (1999) Insertion of a synthetic peptide into a recombinant protein framework; a protein biosensor. J Am Chem Soc 121:1100–1101Google Scholar
  47. 47.
    Tolbert TJ, Wong C-H (2002) New methods for proteomic research: preparation of proteins with N-terminal cysteines for labeling and conjugation. Angew Chem Int Ed Engl 41:2171–2174PubMedGoogle Scholar
  48. 48.
    Liu D, Xu R, Dutta K, Cowburn D (2008) N-terminal cysteinyl proteins can be prepared using thrombin cleavage. FEBS Lett 582:1163–1167PubMedGoogle Scholar
  49. 49.
    Hauser PS, Ryan RO (2007) Expressed protein ligation using an N-terminal cysteine containing fragment generated in vivo from a pelB fusion protein. Protein Expr Purif 54:227–233PubMedGoogle Scholar
  50. 50.
    Dalbey RE, Lively MO, Bron S, van Dijl JM (1997) The chemistry and enzymology of the type I signal peptidases. Protein Sci 6:1129–1138PubMedGoogle Scholar
  51. 51.
    Paetzel M, Dalbey RE, Strynadka NC (2002) Crystal structure of a bacterial signal peptidase apoenzyme: implications for signal peptide binding and the Ser–Lys dyad mechanism. J Biol Chem 277:9512–9519PubMedGoogle Scholar
  52. 52.
    Southworth MW, Amaya K, Evans TC, Xu MQ, Perler FB (1999) Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein. Biotechniques 27:110–114, 116, 118–120Google Scholar
  53. 53.
    Mathys S, Evans TC, Chute IC, Wu H, Chong S, Benner J, Liu XQ, Xu MQ (1999) Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation. Gene 231:1–13PubMedGoogle Scholar
  54. 54.
    Watzke A, Gutierrez-Rodriguez M, Kohn M, Wacker R, Schroeder H, Breinbauer R, Kuhlmann J, Alexandrov K, Niemeyer CM, Goody RS, Waldmann H (2006) A generic building block for C- and N-terminal protein-labeling and protein-immobilization. Bioorg Med Chem 14:6288–6306PubMedGoogle Scholar
  55. 55.
    Watzke A, Kohn M, Gutierrez-Rodriguez M, Wacker R, Schroder H, Breinbauer R, Kuhlmann J, Alexandrov K, Niemeyer CM, Goody RS, Waldmann H (2006) Site-selective protein immobilization by staudinger ligation. Angew Chem Int Ed Engl 45:1408–1412PubMedGoogle Scholar
  56. 56.
    Lin PC, Ueng SH, Tseng MC, Ko JL, Huang KT, Yu SC, Adak AK, Chen YJ, Lin CC (2006) Site-specific protein modification through Cu(I)-catalyzed 1, 2, 3-triazole formation and its implementation in protein microarray fabrication. Angew Chem Int Ed Engl 45:4286–4290PubMedGoogle Scholar
  57. 57.
    Kalia J, Raines RT (2006) Reactivity of intein thioesters: appending a functional group to a protein. Chembiochem 7:1375–1383PubMedGoogle Scholar
  58. 58.
    Camarero JA, Kwon Y, Coleman MA (2004) Chemoselective attachment of biologically active proteins to surfaces by expressed protein ligation and its application for “protein chip” fabrication. J Am Chem Soc 126:14730–14731PubMedGoogle Scholar
  59. 59.
    Girish A, Sun H, Yeo DS, Chen GY, Chua TK, Yao SQ (2005) Site-specific immobilization of proteins in a microarray using intein-mediated protein splicing. Bioorg Med Chem Lett 15:2447–2451PubMedGoogle Scholar
  60. 60.
    Camarero JA, Muir TW (1999) Biosynthesis of a head-to-tail cyclized protein with improved biological activity. J Am Chem Soc 121:5597–5598Google Scholar
  61. 61.
    Kimura RH, Tran AT, Camarero JA (2006) Biosynthesis of the cyclotide Kalata B1 by using protein splicing. Angew Chem Int Ed Engl 45:973–976PubMedGoogle Scholar
  62. 62.
    Camarero JA, Kimura RH, Woo YH, Shekhtman A, Cantor J (2007) Biosynthesis of a fully functional cyclotide inside living bacterial cells. Chembiochem 8:1363–1366PubMedGoogle Scholar
  63. 63.
    Valiyaveetil FI, Leonetti M, Muir TW, Mackinnon R (2006) Ion selectivity in a semisynthetic K+ channel locked in the conductive conformation. Science 314:1004–1007PubMedGoogle Scholar
  64. 64.
    Scheibner KA, Zhang Z, Cole PA (2003) Merging fluorescence resonance energy transfer and expressed protein ligation to analyze protein–protein interactions. Anal Biochem 317:226–232PubMedGoogle Scholar
  65. 65.
    Muralidharan V, Cho J, Trester-Zedlitz M, Kowalik L, Chait BT, Raleigh DP, Muir TW (2004) Domain-specific incorporation of noninvasive optical probes into recombinant proteins. J Am Chem Soc 126:14004–14012PubMedGoogle Scholar
  66. 66.
    Romanelli A, Shekhtman A, Cowburn D, Muir TW (2004) Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction. Proc Natl Acad Sci USA 101:6397–6402Google Scholar
  67. 67.
    Anderson LL, Marshall GR, Crocker E, Smith SO, Baranski TJ (2005) Motion of carboxyl terminus of Galpha is restricted upon G protein activation. A solution NMR study using semisynthetic Galpha subunits. J Biol Chem 280:31019–31026Google Scholar
  68. 68.
    Vitali F, Henning A, Oberstrass FC, Hargous Y, Auweter SD, Erat M, Allain FH (2006) Structure of the two most C-terminal RNA recognition motifs of PTB using segmental isotope labeling. EMBO J 25:150–162PubMedGoogle Scholar
  69. 69.
    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 375:151–164PubMedGoogle Scholar
  70. 70.
    Goody RS, Durek T, Waldmann H, Brunsveld L, Alexandrov K (2005) Application of protein semisynthesis for the construction of functionalized posttranslationally modified rab GTPases. Methods Enzymol 403:29–42PubMedGoogle Scholar
  71. 71.
    Brunsveld L, Kuhlmann J, Alexandrov K, Wittinghofer A, Goody RS, Waldmann H (2006) Lipidated ras and rab peptides and proteins-synthesis, structure, and function. Angew Chem Int Ed Engl 45:6622–6646PubMedGoogle Scholar
  72. 72.
    Reuther G, Tan KT, Vogel A, Nowak C, Arnold K, Kuhlmann J, Waldmann H, Huster D (2006) The lipidated membrane anchor of full length N-Ras protein shows an extensive dynamics as revealed by solid-state NMR spectroscopy. J Am Chem Soc 128:13840–13846PubMedGoogle Scholar
  73. 73.
    Camarero JA (2008) Recent developments in the site-specific immobilization of proteins onto solid supports. Biopolymers 90:450–458PubMedGoogle Scholar
  74. 74.
    Lesaicherre ML, Uttamchandani M, Chen GY, Yao SQ (2002) Developing site-specific immobilization strategies of peptides in a microarray. Bioorg Med Chem Lett 12:2079–2083PubMedGoogle Scholar
  75. 75.
    Govindaraju T, Jonkheijm P, Gogolin L, Schroeder H, Becker CF, Niemeyer CM, Waldmann H (2008) Surface immobilization of biomolecules by click sulfonamide reaction. Chem Commun (Camb) 32:3723–3725Google Scholar
  76. 76.
    Camarero JA, Kwon Y (2008) Traceless and site-specific attachment of proteins onto solid supports. Int J Pept Res Ther 14:351–357Google Scholar
  77. 77.
    Lesaicherre ML, Lue RY, Chen GY, Zhu Q, Yao SQ (2002) Intein-mediated biotinylation of proteins and its application in a protein microarray. J Am Chem Soc 124:8768–8769PubMedGoogle Scholar
  78. 78.
    Lue RY, Chen GY, Hu Y, Zhu Q, Yao SQ (2004) Versatile protein biotinylation strategies for potential high-throughput proteomics. J Am Chem Soc 126:1055–1062PubMedGoogle Scholar
  79. 79.
    Holland-Nell K, Beck-Sickinger AG (2007) Specifically immobilised Aldo/Keto reductase AKR1A1 shows a dramatic increase in activity relative to the randomly immobilised enzyme. Chembiochem 8:1071–1076PubMedGoogle Scholar
  80. 80.
    Hruby VJ, Al-Obeidi F (1990) Emerging approaches in the molecular design of receptor-selective peptide ligands: conformational, topographical and dynamic considerations. J Biochem 268:249–262Google Scholar
  81. 81.
    Rizo J, Gierasch LM (1992) Constrained peptides: models of bioactive peptides and protein substructures. Annu Rev Biochem 61:387–418PubMedGoogle Scholar
  82. 82.
    Camarero JA, Muir TW (1997) Chemoselective backbone cyclization of unprotected peptides. J Chem Soc Chem Comm 27:1369–1370Google Scholar
  83. 83.
    Zhang L, Tam JP (1997) Synthesis and application of unprotected cyclic peptides as building blocks for peptide dendrimers. J Am Chem Soc 119:2363–2370Google Scholar
  84. 84.
    Camarero JA, Pavel J, Muir TW (1998) Chemical synthesis of a circular protein domain: evidence for folding-assisted cyclization. Angew Chem Int Ed 37:347–349Google Scholar
  85. 85.
    Shao Y, Lu WY, Kent SBH (1998) A novel method to synthesize cyclic peptides. Tetrahedron Lett 39:3911–3914Google Scholar
  86. 86.
    Evans TC, Benner J, Xu M-Q (1999) The cyclization and polymerization of bacterially expressed proteins using modified sef-splicing inteins. J Biol Chem 274:18359–18363PubMedGoogle Scholar
  87. 87.
    Camarero JA, Fushman D, Sato S, Giriat I, Cowburn D, Raleigh DP, Muir TW (2001) Rescuing a destabilized protein fold through backbone cyclization. J Mol Biol 308:1045–1062PubMedGoogle Scholar
  88. 88.
    Craik DJ, Daly NL, Bond T, Waine C (1999) Plant cyclotides: a unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J Mol Biol 294:1327–1336PubMedGoogle Scholar
  89. 89.
    Greenwood KP, Daly NL, Brown DL, Stow JL, Craik DJ (2007) The cyclic cystine knot miniprotein MCoTI-II is internalized into cells by macropinocytosis. Int J Biochem Cell Biol 39:2252–2264PubMedGoogle Scholar
  90. 90.
    Craik DJ, Simonsen S, Daly NL (2002) The cyclotides: novel macrocyclic peptides as scaffolds in drug design. Curr Opin Drug Discov Devel 5:251–260PubMedGoogle Scholar
  91. 91.
    Salzmann M, Pervushin K, Wider G, Senn H, Wuthrich K (1998) TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc Natl Acad Sci USA 95:13585–13590PubMedGoogle Scholar
  92. 92.
    Xu R, Ayers B, Cowburn D, Muir TW (1999) Chemical ligation of folded recombinant proteins; segmental isotopic labeling of domains for NMR studies. Proc Natl Acad Sci USA 96:388–393PubMedGoogle Scholar
  93. 93.
    Zhao W, Zhang Y, Cui C, Li Q, Wang J (2008) An efficient on-column expressed protein ligation strategy: application to segmental triple labeling of human apolipoprotein E3. Protein Sci 17:736–747PubMedGoogle Scholar
  94. 94.
    Tatulian SA, Qin S, Pande AH, He X (2005) Positioning membrane proteins by novel protein engineering and biophysical approaches. J Mol Biol 351:939–947PubMedGoogle Scholar
  95. 95.
    Flavell RR, Kothari P, Bar-Dagan M, Synan M, Vallabhajosula S, Friedman JM, Muir TW, Ceccarini G (2008) Site-specific (18)F-labeling of the protein hormone leptin using a general two-step ligation procedure. J Am Chem Soc 130:9106–9112PubMedGoogle Scholar
  96. 96.
    Flavell RR, Huse M, Goger M, Trester-Zedlitz M, Kuriyan J, Muir TW (2002) Efficient semisynthesis of a tetraphosphorylated analogue of the Type I TGFbeta receptor. Org Lett 4:165–168PubMedGoogle Scholar
  97. 97.
    Ottesen JJ, Huse M, Sekedat MD, Muir TW (2004) Semisynthesis of phosphovariants of Smad2 reveals a substrate preference of the activated T beta RI kinase. Biochemistry 43:5698–5706PubMedGoogle Scholar
  98. 98.
    Wu JW, Hu M, Chai J, Seoane J, Huse M, Li C, Rigotti DJ, Kyin S, Muir TW, Fairman R, Massague J, Shi Y (2001) Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling. Mol Cell 8:1277–1289PubMedGoogle Scholar
  99. 99.
    Huse M, Muir TW, Xu L, Chen YG, Kuriyan J, Massague J (2001) The TGF beta receptor activation process: an inhibitor- to substrate-binding switch. Mol Cell 8:671–682PubMedGoogle Scholar
  100. 100.
    Qin BY, Lam SS, Correia JJ, Lin K (2002) Smad3 allostery links TGF-beta receptor kinase activation to transcriptional control. Genes Dev 16:1950–1963PubMedGoogle Scholar
  101. 101.
    Chacko BM, Qin BY, Tiwari A, Shi G, Lam S, Hayward LJ, De Caestecker M, Lin K (2004) Structural basis of heteromeric smad protein assembly in TGF-beta signaling. Mol Cell 15:813–823PubMedGoogle Scholar
  102. 102.
    Durek T, Alexandrov K, Goody RS, Hildebrand A, Heinemann I, Waldmann H (2004) Synthesis of fluorescently labeled mono- and diprenylated Rab7 GTPase. J Am Chem Soc 126:16368–16378PubMedGoogle Scholar
  103. 103.
    Brunsveld L, Watzke A, Durek T, Alexandrov K, Goody RS, Waldmann H (2005) Synthesis of functionalized rab GTPases by a combination of solution- or solid-phase lipopeptide synthesis with expressed protein ligation. Chemistry 11:2756–2772PubMedGoogle Scholar
  104. 104.
    Rak A, Pylypenko O, Durek T, Watzke A, Kushnir S, Brunsveld L, Waldmann H, Goody RS, Alexandrov K (2003) Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase. Science 302:646–650PubMedGoogle Scholar
  105. 105.
    Pylypenko O, Rak A, Durek T, Kushnir S, Dursina BE, Thomae NH, Constantinescu AT, Brunsveld L, Watzke A, Waldmann H, Goody RS, Alexandrov K (2006) Structure of doubly prenylated Ypt1:GDI complex and the mechanism of GDI-mediated Rab recycling. EMBO J 25:13–23PubMedGoogle Scholar
  106. 106.
    Guo Z, Wu YW, Das D, Delon C, Cramer J, Yu S, Thuns S, Lupilova N, Waldmann H, Brunsveld L, Goody RS, Alexandrov K, Blankenfeldt W (2008) Structures of RabGGTase-substrate/product complexes provide insights into the evolution of protein prenylation. EMBO J 27:2444–2456PubMedGoogle Scholar
  107. 107.
    Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693–705PubMedGoogle Scholar
  108. 108.
    Shogren-Knaak MA, Fry CJ, Peterson CL (2003) A native peptide ligation strategy for deciphering nucleosomal histone modifications. J Biol Chem 278:15744–15748PubMedGoogle Scholar
  109. 109.
    Shogren-Knaak M, Ishii H, Sun JM, Pazin MJ, Davie JR, Peterson CL (2006) Histone H4–K16 acetylation controls chromatin structure and protein interactions. Science 311:844–847PubMedGoogle Scholar
  110. 110.
    He S, Bauman D, Davis JS, Loyola A, Nishioka K, Gronlund JL, Reinberg D, Meng F, Kelleher N, McCafferty DG (2003) Facile synthesis of site-specifically acetylated and methylated histone proteins: reagents for evaluation of the histone code hypothesis. Proc Natl Acad Sci USA 100:12033–12038PubMedGoogle Scholar
  111. 111.
    Chatterjee C, McGinty RK, Pellois JP, Muir TW (2007) Auxiliary-mediated site-specific peptide ubiquitylation. Angew Chem Int Ed Engl 46:2814–2818PubMedGoogle Scholar
  112. 112.
    McGinty RK, Kim J, Chatterjee C, Roeder RG, Muir TW (2008) Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453:812–816PubMedGoogle Scholar
  113. 113.
    Hojo H, Nakahara Y (2007) Recent progress in the field of glycopeptide synthesis. Biopolymers 88:308–324PubMedGoogle Scholar
  114. 114.
    Marcaurelle LA, Mizoue LS, Wilken J, Oldham L, Kent SB, Handel TM, Bertozzi CR (2001) Chemical synthesis of lymphotactin: a glycosylated chemokine with a C-terminal mucin-like domain. Chemistry 7:1129–1132PubMedGoogle Scholar
  115. 115.
    Hackenberger CP, Friel CT, Radford SE, Imperiali B (2005) Semisynthesis of a glycosylated Im7 analogue for protein folding studies. J Am Chem Soc 127:12882–12889PubMedGoogle Scholar
  116. 116.
    Macmillan D, Bertozzi CR (2004) Modular assembly of glycoproteins: towards the synthesis of GlyCAM-1 by using expressed protein ligation. Angew Chem Int Ed Engl 43:1355–1359PubMedGoogle Scholar
  117. 117.
    Valiyaveetil FI, Sekedat M, MacKinnon R, Muir TW (2006) Structural and functional consequences of an amide-to-ester substitution in the selectivity filter of a potassium channel. J Am Chem Soc 128:11591–11599PubMedGoogle Scholar
  118. 118.
    Schwarzer D, Cole PA (2005) Protein semisynthesis and expressed protein ligation: chasing a protein’s tail. Curr Opin Chem Biol 9:561–569PubMedGoogle Scholar
  119. 119.
    Algire MA, Maag D, Lorsch JR (2005) Pi release from eIF2, not GTP hydrolysis, is the step controlled by start-site selection during eukaryotic translation initiation. Mol Cell 20:251–262Google Scholar
  120. 120.
    Maag D, Fekete CA, Gryczynski Z, Lorsch JR (2005) A conformational change in the eukaryotic translation preinitiation complex and release of eIF1 signal recognition of the start codon. Mol Cell 17:265–275PubMedGoogle Scholar
  121. 121.
    Szewczuk LM, Tarrant MK, Sample V, Drury WJ 3rd, Zhang J, Cole PA (2008) Analysis of serotonin N-acetyltransferase regulation in vitro and in live cells using protein semisynthesis. Biochemistry 47:10407–10419PubMedGoogle Scholar
  122. 122.
    Xie N, Elangwe EN, Asher S, Zheng YG (2009) A dual-mode fluorescence strategy for screening HAT modulators. Bioconjug Chem 20:360–366PubMedGoogle Scholar
  123. 123.
    Woo Y-H, Camarero JA (2006) Interfacing ‘Hard’ and ‘Soft’ matter with exquisite chemical control. Curr Nanosci 2:93–103Google Scholar
  124. 124.
    Becker CF, Marsac Y, Hazarika P, Moser J, Goody RS, Niemeyer CM (2007) Functional immobilization of the small GTPase Rab6A on DNA-Gold nanoparticles by using a site-specifically attached poly(ethylene glycol) linker and thiol place-exchange reaction. Chembiochem 8:32–36PubMedGoogle Scholar
  125. 125.
    Craik DJ, Cemazar M, Daly NL (2006) The cyclotides and related macrocyclic peptides as scaffolds in drug design. Curr Opin Drug Discov Dev 9:251–260Google Scholar
  126. 126.
    Paulick MG, Wise AR, Forstner MB, Groves JT, Bertozzi CR (2007) Synthetic analogues of glycosylphosphatidylinositol-anchored proteins and their behavior in supported lipid bilayers. J Am Chem Soc 129:11543–11550PubMedGoogle Scholar

Copyright information

© Birkhäuser Verlag, Basel/Switzerland 2009

Authors and Affiliations

  1. 1.Department of Pharmacology and Pharmaceutical Sciences, School of PharmacyUniversity of Southern CaliforniaLos AngelesUSA

Personalised recommendations