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Convergent protein synthesis
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Convergent protein synthesis Giulio Casi and Donald Hilvert Methods for the chemical synthesis of proteins have advanced considerably over the past decade. In many instances, laboratory synthesis can now be considered a viable alternative to ribosomal biosynthesis, especially when custom modifications of a protein are desired; chemical approaches guarantee virtually unlimited and tunable variation of the covalent structure of a polypeptide. Addresses Laboratorium fu¨r Organische Chemie, Swiss Federal Institute of Technology, ETH-Ho¨nggerberg, CH-8093 Zu¨rich, Switzerland e-mail: [email protected]
Current Opinion in Structural Biology 2003, 13:589–594 This review comes from a themed issue on Biophysical methods Edited by Brian T Chait and Keith Moffat 0959-440X/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved.
methods for coupling peptide segments (Figure 1). To that end, two basic problems must be solved. First, the C terminus of the acceptor fragment must be selectively activated, while at the same time minimizing the tendency of activated acyl derivatives to racemize. Second, a donor fragment with compatible reactivity must be found. Thioesters have proven to be broadly useful acceptor electrophiles in protein synthesis [4], although glycoaldehydes, formyl groups and perthioesters have also been employed [5]. C-Terminal peptide thioesters are readily prepared by SPPS [6–9]. They can also be produced recombinantly, using intein-mediated protein splicing [10]. Inteins are self-splicing proteins that promote a series of intramolecular reactions leading to their posttranslational excision from a larger precursor protein and the concomitant ligation of the flanking peptide segments, called exteins. Modified inteins have been designed that undergo partial self-splicing and allow efficient trapping of recombinant N-terminal exteins with thiols.
DOI 10.1016/j.sbi.2003.09.008
Abbreviations EPL expressed protein ligation Epo erythropoietin GFP green fluorescent protein NCL native chemical ligation PTD protein transduction domain SPPS solid-phase peptide synthesis
When peptide thioesters are mixed with a second peptide containing an N-terminal cysteine as the reactive donor fragment, they undergo rapid transthioesterification. The resulting thioester intermediate rearranges spontaneously and irreversibly by acyl migration, from sulfur to the aamine, to link the two peptides together via a ‘native’ amide bond (Figure 1b) [11,12]. This process, termed ‘native chemical ligation’ (NCL), has been widely applied to the synthesis of numerous moderately sized peptides and proteins [4].
Introduction The chemical synthesis of proteins has traditionally involved the sequential coupling of amino acids on a solid support. Decades of optimization have made stepwise solid-phase peptide synthesis (SPPS) a routine and reliable tool for preparing peptides up to 20–30 amino acids long [1]. In favorable cases, even longer polymers can be produced, as illustrated by the synthesis of ribonuclease A (124 amino acids) [2] and HIV protease (99 amino acids) [3]. In general, however, yields decrease with increasing length of the polypeptide and removal of the impurities that accumulate during the synthesis of longer polymers can often require heroic effort. Convergent assembly of proteins from peptide fragments largely overcomes the limitations of a purely stepwise approach and significantly extends the size of molecules that can be prepared in the laboratory [4,5].
Chemoselective ligations Convergent strategies for the synthesis of proteins depend directly on the availability of chemoselective www.current-opinion.com
The NCL strategy is general and quite robust. In addition to cysteine, many other nucleophilic residues can mediate chemical ligation, including homocysteine [13], selenocysteine [14–16], selenohomocysteine [17] and histidine [18]. An even broader range of formal ligation sites can be accessed by reducing the C–S [19] or C–Se [20] bond after the amide bond has formed, or by using removable sulfurcontaining auxiliaries to facilitate fragment condensation [21,22,23]. In some cases, it is possible to perform ligations with donor fragments lacking a nucleophilic sidechain at their N terminus, for example when the reaction is carried out under conditions favoring a folded state that holds the reactive amine proximal to the thioester [24]. When larger proteins are desired, serial ligations of multiple peptide segments can be performed, although special protecting groups or orthogonal coupling methods may be required. The Staudinger ligation, which forms an amide bond from an azide and a specifically functionalized phosphine (Figure 1b), is an example of the latter. Alkyl Current Opinion in Structural Biology 2003, 13:589–594
590 Biophysical methods
Figure 1
through SPPS alone, as shown by the semisynthesis of analogs of the b0 subunit of Escherichia coli RNA polymerase, which contains 1407 amino acid residues [30]. EPL also makes possible segmental labeling of large proteins [31], which is of considerable benefit to NMR structural studies.
(a)
+
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Over the past year, chemoselective ligations have been exploited in many different ways. They have been used to prepare membrane proteins [32], enzymes containing unnatural amino acids for detailed investigations of chemical mechanism [33], and proteins modified with biophysical probes to explore protein folding and dynamics, and interactions between protein domains [17,34]. Also, a selectively lipidated protein was synthesized for studies of post-translational prenylation [35]. The fact that the number and nature of modifications are determined completely by the synthetic protocol, affording homogenously modified proteins, contrasts with cellular post-translational processes which often yield heterogeneous mixtures.
azides react with triarylphosphines to form aza-ylides [25]. If the aza-ylide is produced adjacent to an ester or thioester, intramolecular acyl transfer occurs efficiently to afford a new amide bond. Subsequent hydrolysis liberates the ligated peptide plus phosphine oxide. ‘Traceless’ versions of this method have been developed that leave no residual atoms from the triarylphosphine prosthetic group in the product [26–28]. The great advantage of the Staudinger ligation is that the donor fragment does not require an N-terminal residue with a nucleophilic sidechain. Thus, it can be applied in combination with NCL to generate larger proteins, as illustrated by the preparation of an isotopically labeled version of the enzyme ribonuclease A [29].
Importantly, one need not adhere to nature’s constraints, as shown by the preparation of an artificial variant of erythropoietin (Epo), a glycoprotein hormone that normally regulates the proliferation, differentiation and maturation of erythroid cells. The synthesis of this 166 amino acid protein, covalently linked to two negatively charged, branched and monodisperse synthetic polymers rather than to natural carbohydrates, is a tour de force [36]. Two peptide–polymer conjugates were initially constructed by a chemoselective oxime-forming ligation reaction between an aminoxy group on the polymer and a ketone-bearing lysine on the respective peptides; these were then coupled with two other peptide segments using thioligation methods (Figure 2). After the first two fragment condensations, the cysteine residues involved were alkylated with bromoacetic acid to provide an amino acid at the ligation sites that is electronically and sterically similar to the glutamate residue found at the corresponding positions in Epo. This procedure provided more than 100 mg of polymer-modified Epo. The artificial hormone was shown to have potent haematopoietic activity in cell and animal assays, with notably prolonged duration of action in vivo relative to natural Epo. The design and synthesis of this 51 kDa macromolecule impressively illustrates how modern chemoselective ligation strategies can provide precise control over covalent structure and molecular function. In principle, the properties of many proteins of pharmaceutical interest could be similarly fine-tuned.
In many instances, one or more of the peptide segments used to assemble a protein is produced recombinantly and the assembly process is then referred to as ‘expressed protein ligation’ (EPL) [10]. Facile production of recombinant fragments has the decided advantage that much larger molecules can be tackled than are accessible
The same ligation strategies that have proven so efficacious in constructing polypeptides can also be expected to find wide application in the construction of artificial polymers based on non-natural backbones. As a case in point, b-peptides are efficiently coupled using thioligation chemistry [37]. Chimeric proteins, in which nonpeptidic
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P R R Current Opinion in Structural Biology
Chemoselective ligations. (a) Schematic representation of peptide fragment condensation. (b) NCLs and Staudinger ligations are chemoselective and orthogonal ways of forming amide bonds between peptide segments. P1 and P2 represent the N- and C-terminal peptides, respectively.
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Figure 2
(Acm)S SR
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(ii) Three successive ligations H N
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Current Opinion in Structural Biology
Synthesis of a synthetic erythropoiesis protein (SEP). Four unprotected peptide fragments were produced by SPPS. Two of the fragments, SEP (1–32) and SEP (117–166), which contain nonstandard Ne-levulinyl lysine residues, were subsequently modified via an oxime-forming reaction with an anionic, branched and monodisperse synthetic polymer (i). The mainchain was then assembled from the C to the N terminus by three successive NCLs (ii). After the first two condensations, the cysteine residues at positions 89 and 117 were alkylated with bromoacetic acid to mimic the glutamate residues found at the corresponding positions in Epo. The full-length peptide–polymer conjugate was folded under oxidative conditions to give the biologically active artificial hormone (iii). The peptide is shown as a ribbon diagram (blue) based on the coordinates for Epo (PDB entry 1BUY) and the branched polymers (red) are scaled to represent the approximate hydrodynamic volume of SEP [36].
elements are incorporated into the peptide backbone, have also been prepared, including a ribonuclease derivative containing a b-peptide reverse turn [38]. Ready access to other non-natural polymers of defined length and composition is likely to afford many exciting opportunities in medicine, material science and catalyst design [39].
Protein semisynthesis in vivo The burgeoning revolution in proteomics is fueling the need for proteins with tailored modifications. Designer proteins are facilitating efforts to characterize the movement, interactions and chemical microenvironment of macromolecules inside living cells [40], as well as the construction of protein microarrays for defined assays in vitro [41]. A common biological approach is to create recombinant fusion proteins, in which the protein of interest is covalently linked to another protein, such as glutathione S-transferase or green fluorescent protein (GFP) [42,43], which provide convenient handles for purification, targeting or biophysical investigation. If www.current-opinion.com
proteins contain uniquely reactive sites, they can also be selectively modified with cell-permeable fluorophores or other probes [44,45]. Alternatively, suppressor tRNAs and engineered tRNA synthetases are being developed that may eventually enable the site-selective incorporation of virtually any nonstandard amino acid into any protein in the proteome [46]. Each approach has advantages and disadvantages, and the method of choice will ultimately depend on the envisaged application. Convergent protein synthesis has considerable potential as a complementary approach for engineering proteins intracellularly. The first steps in this direction have already been taken, with trans-splicing reactions mediated by inteins playing a key role. In protein trans-splicing, the intein is split into two components. These must assemble to initiate the autocatalytic process that leads to excision of the intein fragments and ligation of the flanking N- and C-terminal domains. Such systems have already been modified to produce cyclic peptides and proteins that Current Opinion in Structural Biology 2003, 13:589–594
592 Biophysical methods
exhibit enhanced thermodynamic stability and increased resistance to exo-peptidase digestion in vivo [47–51,52]. The cyclic products can also be screened directly for antibiotic activity or other interesting properties. For example, a split intein has been engineered into a retroviral expression system for the intracellular delivery of random libraries of cyclic peptides to human cells [53]. Coupled with a functional genetic screen, this method afforded cyclic pentapeptides that selectively block interleukin-4 signaling. As shown in Figure 3, protein trans-splicing also provides a means of selectively labeling recombinant proteins inside a cell. The target molecule is biosynthesized intracellularly with one-half of the split intein (IN) fused to its C terminus. The other half of the intein (IC) is fused to the molecular probe and linked via a disulfide bond to a protein transduction domain (PTD) peptide. When this semisynthetic construct is added to the medium, the PTD peptide carries it into the cell, where the disulfide bond is cleaved, the split inteins assemble and protein Figure 3
PTD Probe
C
IC Delivery and reduction DNA PTD Expression
Protein
C
IC
IN
Probe
Complementation
Protein
IN IC
C
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splicing occurs to link the target protein to the probe. The feasibility of this scheme has been demonstrated through semisynthesis of a GFP variant bearing a C-terminal FLAG epitope [54]. In the future, however, it should be possible to exploit trans-splicing to couple many peptides and modified cysteine derivatives to proteins in vivo. The splicing process depicted in Figure 3 is triggered by the exogenous addition of one component to cells already producing the other. Consequently, temporal control of protein modification is inherent to the system. It may also be possible to regulate protein trans-splicing inside living cells using small molecules. In fact, an artificial heterodimeric split intein has been engineered that dimerizes only upon binding of a small bidentate ligand [55]. This system has considerable promise for in vivo applications [56].
Perspectives Versatile methods for chemoselectively coupling peptides have provided a practical basis for constructing proteins in a general and modular fashion. Methodological improvements that further increase the efficiency of existing ligation chemistry, afford orthogonal ways of linking peptide segments with an extended range of permissible coupling sites, and enable synthetic manipulations in in vivo settings will make chemical synthesis of proteins an increasingly attractive alternative to purely biological production. The key advantages of the chemical approach are an exacting control over covalent structure and the ability to explore structural realms outside nature’s normal scope. In sum, chemical synthesis is a powerful enabling technology. Chemists and biologists interested in developing new materials or exploring problems in basic science — including the molecular basis of protein function and the roles proteins play in complex biochemical pathways within the cell — stand to benefit enormously from ready access to synthetic proteins with tailored properties. As we look beyond today’s horizons, toward novel nonpeptide polymeric worlds created by analogous strategies and with the same control as natural polypeptides, exciting and unexpected discoveries are sure to be made.
Acknowledgements
IN
Protein splicing
IC
Protein
C
Probe
We thank the ETH Zu¨ rich and Novartis Pharma for financial support, and Ken Woycechowsky for helpful discussions. GC is grateful to the Roche Research Foundation for a doctoral fellowship.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
Current Opinion in Structural Biology
Protein trans-splicing strategy for modifying recombinant proteins in vivo with an exogenously added probe. The two halves of the split intein are designated IC and IN; PTD is a protein transduction domain peptide that enables cellular uptake of the probe molecule. C is the cysteine involved in protein splicing and used to link the PTD to the IC probe. Current Opinion in Structural Biology 2003, 13:589–594
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Muir TW: Semisynthesis of proteins by expressed protein ligation. Annu Rev Biochem 2003, 72:249-289. thorough review summarizes the development and application of methods, and discusses their potential in the field of proteomics.
11. Wieland T, Bokelmann E, Bauer L, Lang HU, Lau H: Polypeptide synthesis. VIII. Formation of sulfur containing peptides by the intramolecular migration of aminoacyl groups. Liebigs Ann Chem 1953, 583:129-149. 12. Dawson PE, Muir TW, Clark-Lewis I, Kent SBH: Synthesis of proteins by native chemical ligation. Science 1994, 266:776-779. 13. Tam JP, Yu QT: Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones. Biopolymers 1998, 46:319-327. 14. Gieselman MD, Xie L, van der Donk WA: Synthesis of a selenocysteine-containing peptide by native chemical ligation. Org Lett 2001, 3:1331-1334. 15. Hondal RJ, Nilsson BL, Raines RT: Selenocysteine in native chemical ligation and expressed protein ligation. J Am Chem Soc 2001, 123:5140-5141. 16. Quaderer R, Sewing A, Hilvert D: Selenocysteine-mediated native chemical ligation. Helv Chim Acta 2001, 84:1197-1206. 17. Roelfes G, Hilvert D: Incorporation of selenomethionine into proteins through selenohomocysteine-mediated ligation. Angew Chem Int Ed Engl 2003, 42:2275-2277. Selenohomocysteine is shown to be a viable substitute for cysteine in NCLs. Methylation of the selenol following fragment condensation yields a selenomethionine at the coupling site, which is a useful spectroscopic probe of local protein structure and dynamics, as illustrated for a synthetic variant of bovine pancreatic polypeptide. 18. Zhang LS, Tam JP: Orthogonal coupling of unprotected peptide segments through histidyl amino terminus. Tetrahedron Lett 1997, 38:3-6. 19. Yan LZ, Dawson PE: Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J Am Chem Soc 2001, 123:526-533. 20. Quaderer R, Hilvert D: Selenocysteine-mediated backbone cyclization of unprotected peptides followed by alkylation, oxidative elimination or reduction of the selenol. Chem Commun 2002:2620-2621. Like cysteine, N-terminal selenocysteine facilitates cyclization of peptide thioesters. Subsequent reduction, elimination or alkylation of the selenol affords modified cyclic peptides with alanine, dehydroalanine or a nonstandard amino acid at the ligation site. 21. Low DW, Hill MG, Carrasco MR, Kent SBH, Botti P: Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary. Proc Natl Acad Sci USA 2001, 98:6554-6559. www.current-opinion.com
22. Offer J, Boddy CNC, Dawson PE: Extending synthetic access to proteins with a removable acyl transfer auxiliary. J Am Chem Soc 2002, 124:4642-4646. An acid-labile 2-mercaptobenzyl auxiliary is described that can replace cysteine in NCLs. Its utility was demonstrated by the synthesis of a 62 amino acid SH3 domain through ligation at a Lys–Gly sequence. 23. Botti P, Carrasco MR, Kent SBH: Native chemical ligation using removable Na-(1-phenyl-2- mercaptoethyl) auxiliaries. Tetrahedron Lett 2001, 42:1831-1833. 24. Beligere GS, Dawson PE: Conformationally assisted protein ligation using C-terminal thioester peptides. J Am Chem Soc 1999, 121:6332-6333. 25. Staudinger H, Meyer J: New organic compounds of phosphorous. III. Phosphinemethylene derivatives and phosphinimines. Helv Chim Acta 1919, 2:635-646. 26. Nilsson BL, Kiessling LL, Raines RT: Staudinger ligation: a peptide from a thioester and azide. Org Lett 2000, 2:1939-1941. 27. Saxon E, Armstrong JI, Bertozzi CR: A ‘traceless’ Staudinger ligation for the chemoselective synthesis of amide bonds. Org Lett 2000, 2:2141-2143. 28. Nilsson BL, Kiessling LL, Raines RT: High-yielding Staudinger ligation of a phosphinothioester and azide to form a peptide. Org Lett 2001, 3:9-12. 29. Nilsson BL, Hondal RJ, Soellner MB, Raines RT: Protein assembly by orthogonal chemical ligation methods. J Am Chem Soc 2003, 125:5268-5269. This article describes the synthesis of ribonuclease A from three fragments, using sequential Staudinger and native chemical ligations. 30. Mukhopadhyay J, Kapanidis AN, Mekler V, Kortkhonjia E, Ebright YW, Ebright RH: Translocation of r70 with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA. Cell 2001, 106:453-463. 31. Xu R, Ayers B, Cowburn D, Muir TW: Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies. Proc Natl Acad Sci USA 1999, 96:388-393. 32. Valiyaveetil FI, MacKinnon R, Muir TW: Semisynthesis and folding of the potassium channel KcsA. J Am Chem Soc 2002, 124:9113-9120. EPL was successfully exploited to synthesize a truncated version of the KcsA potassium channel, which was subsequently incorporated into lipid vesicles. This study highlights the potential utility of ligation strategies for the (semi)synthesis of integral membrane proteins. 33. Kienho¨ fer A, Kast P, Hilvert D: Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donor. J Am Chem Soc 2003, 125:3206-3207. EPL was used to prepare a chorismate mutase variant in which an active site arginine was replaced by an isosteric but neutral citrulline. The resulting dramatic loss of catalytic activity, accompanied by relatively modest effects on substrate and inhibitor binding, provides evidence for selective electrostatic stabilization of the pericyclic transition state. 34. Camarero JA, Shekhtman A, Campbell EA, Chlenov M, Gruber TM, Bryant DA, Darst SA, Cowburn D, Muir TW: Autoregulation of a bacterial r factor explored by using segmental isotopic labeling and NMR. Proc Natl Acad Sci USA 2002, 99:8536-8541. NMR spectroscopic studies of a bacterial s factor, which was prepared in segmentally labeled form by EPL, argue against a high-affinity intramolecular interaction between two regions of this protein, previously believed to be responsible for autoinhibition of its DNA-binding function. 35. Alexandrov K, Heinemann I, Durek T, Sidorovitch V, Goody RS, Waldmann H: Intein-mediated synthesis of geranylgeranylated Rab7 protein in vitro. J Am Chem Soc 2002, 124:5648-5649. A combination of organic synthesis, protein expression and in vitro protein ligation afforded a fluorescent, monoprenylated Rab7 protein for mechanistic studies of the prenylation reaction catalyzed by Rab geranylgeranyltransferase. 36. Kochendoerfer GG, Chen SY, Mao F, Cressman S, Traviglia S, Shao H, Hunter CL, Low DW, Cagle EN, Carnevali M et al.: Design and chemical synthesis of a homogeneous polymer-modified erythropoiesis protein. Science 2003, 299:884-887. The design and total synthesis of ‘synthetic erythropoiesis protein’, a 51 kDa protein–polymer construct, in homogeneous form illustrates the Current Opinion in Structural Biology 2003, 13:589–594
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current frontiers of NCL technology. The artificial hormone displayed potent biological activity in cell and animal assays for erythropoiesis, and had significantly prolonged duration of action in vivo. 37. Kimmerlin T, Seebach D, Hilvert D: Synthesis of b3-peptides and mixed a/b3-peptides by thioligation. Helv Chim Acta 2002, 85:1812-1826. This paper shows that NCL works well for coupling b-peptides to give longer b3 oligomers and mixed a/b3 peptides. 38. Arnold U, Hinderaker MP, Nilsson BL, Huck BR, Gellman SH, Raines RT: Protein prosthesis: a semisynthetic enzyme with a b-peptide reverse turn. J Am Chem Soc 2002, 124:8522-8523. EPL was used to prepare a semisynthetic version of ribonuclease A containing a di-b-peptide unit in place of Asn113–Pro114. The variant retained full catalytic activity and possessed enhanced conformational stability relative to the wild-type enzyme. 39. Gellman SH: Foldamers: a manifesto. Acc Chem Res 1998, 31:173-180. 40. Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P et al.: A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 2000, 403:623-627. 41. MacBeath G, Schreiber SL: Printing proteins as microarrays for high-throughput function determination. Science 2000, 289:1760-1763. 42. Terpe K: Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 2003, 60:523-533. This review provides an up-to-date overview of the most frequently used fusion partners for detecting and purifying recombinant proteins in proteomics research. 43. Tsien RY: The green fluorescent protein. Annu Rev Biochem 1998, 67:509-544. 44. Griffin BA, Adams SR, Tsien RY: Specific covalent labeling of recombinant protein molecules inside live cells. Science 1998, 281:269-272. 45. Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K: A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 2003, 21:86-89. A general method for covalently labeling fusion proteins in vivo is described that relies on selective alkylation of the human DNA repair protein O6-alkylguanine-DNA alkyltranferase with cell-permeable dyes. 46. Wang L, Schultz PG: Expanding the genetic code. Chem Commun 2002:1-11. This article reviews recent progress toward engineering bacteria with expanded genetic repertoires, with emphasis on the development of novel tRNA–codon pairs and aminoacyl-tRNA synthetases for nonstandard amino acids that function orthogonally to those for the 20 natural amino acids. 47. Scott CP, Abel-Santos E, Wall M, Wahnon DC, Benkovic SJ: Production of cyclic peptides and proteins in vivo. Proc Natl Acad Sci USA 1999, 96:13638-13643.
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48. Evans TC, Martin D, Kolly R, Panne D, Sun L, Ghosh I, Chen L, Benner J, Liu XQ, Xu MQ: Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803. J Biol Chem 2000, 275:9091-9094. 49. Camarero JA, Fushman D, Cowburn D, Muir TW: Peptide chemical ligation inside living cells: in vivo generation of a circular protein domain. Bioorg Med Chem 2001, 9:2479-2484. 50. Iwai H, Lingel A, Plu¨ ckthun A: Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J Biol Chem 2001, 276:16548-16554. 51. Scott CP, Abel-Santos E, Jones AD, Benkovic SJ: Structural requirements for the biosynthesis of backbone cyclic peptide libraries. Chem Biol 2001, 8:801-815. 52. Williams NK, Prosselkov P, Liepinsh E, Line I, Sharipo A, Littler DR, Curmi PMG, Otting G, Dixon NE: In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein. J Biol Chem 2002, 277:7790-7798. A synthetic split mini-intein gene was constructed for intracellular cyclization of recombinant proteins produced in E. coli. It was shown to cyclize the N-terminal domain of E. coli DnaB efficiently, without accumulating significant amounts of unprocessed precursor or linear protein, as has been observed with other split intein systems. 53. Kinsella TM, Ohashi CT, Harder AG, Yam GC, Li W, Peelle B, Pali ES, Bennett MK, Molineaux SM, Anderson DA et al.: Retrovirally delivered random cyclic peptide libraries yield inhibitors of interleukin-4 signaling in human B cells. J Biol Chem 2002, 277:37512-37518. A split intein was engineered into a retroviral expression system to facilitate intracellular delivery of a library of random cyclic peptides into human cells. Cyclization of peptides was verified in cell lysates by mass spectrometry and peptides that selectively inhibit germ line epsilon transcription were identified through a functional screen. 54. Giriat I, Muir TW: Protein semi-synthesis in living cells. J Am Chem Soc 2003, 125:7180-7181. A general strategy is presented for exploiting split intein technology to modify proteins with probe molecules in vivo. Its feasibility is demonstrated by attachment of a C-terminal FLAG epitope, introduced exogenously to the growth medium and carried into the cell by a PTD peptide, to intracellular GFP. The method has broad potential for modifying proteins in their natural setting. 55. Mootz HD, Muir TW: Protein splicing triggered by a small molecule. J Am Chem Soc 2002, 124:9044-9045. An approach for regulating protein splicing is outlined. The ability of a small molecule, rapamycin, to heterodimerize proteins FKBP and FRB is exploited to bring together each half of a weakly interacting split intein fused to these two proteins. Protein splicing of the flanking sequences was shown to occur only in the presence of rapamycin. 56. Mootz HD, Blum ES, Tyszkiewicz AB, Muir TW: Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo. J Am Chem Soc 2003, 125:10561-10569. The protein splicing activity of an engineered split intein in mammalian cells was shown to be inducible by exogenous addition of the small molecule rapamycin. Response time, dose dependency and effects of inhibitors were investigated.
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Convergent protein synthesis Giulio Casi and Donald Hilvert Methods for the chemical synthesis of proteins have advanced considerably over the past decade. In many instances, laboratory synthesis can now be considered a viable alternative to ribosomal biosynthesis, especially when custom modifications of a protein are desired; chemical approaches guarantee virtually unlimited and tunable variation of the covalent structure of a polypeptide. Addresses Laboratorium fu¨r Organische Chemie, Swiss Federal Institute of Technology, ETH-Ho¨nggerberg, CH-8093 Zu¨rich, Switzerland e-mail: [email protected]
Current Opinion in Structural Biology 2003, 13:589–594 This review comes from a themed issue on Biophysical methods Edited by Brian T Chait and Keith Moffat 0959-440X/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved.
methods for coupling peptide segments (Figure 1). To that end, two basic problems must be solved. First, the C terminus of the acceptor fragment must be selectively activated, while at the same time minimizing the tendency of activated acyl derivatives to racemize. Second, a donor fragment with compatible reactivity must be found. Thioesters have proven to be broadly useful acceptor electrophiles in protein synthesis [4], although glycoaldehydes, formyl groups and perthioesters have also been employed [5]. C-Terminal peptide thioesters are readily prepared by SPPS [6–9]. They can also be produced recombinantly, using intein-mediated protein splicing [10]. Inteins are self-splicing proteins that promote a series of intramolecular reactions leading to their posttranslational excision from a larger precursor protein and the concomitant ligation of the flanking peptide segments, called exteins. Modified inteins have been designed that undergo partial self-splicing and allow efficient trapping of recombinant N-terminal exteins with thiols.
DOI 10.1016/j.sbi.2003.09.008
Abbreviations EPL expressed protein ligation Epo erythropoietin GFP green fluorescent protein NCL native chemical ligation PTD protein transduction domain SPPS solid-phase peptide synthesis
When peptide thioesters are mixed with a second peptide containing an N-terminal cysteine as the reactive donor fragment, they undergo rapid transthioesterification. The resulting thioester intermediate rearranges spontaneously and irreversibly by acyl migration, from sulfur to the aamine, to link the two peptides together via a ‘native’ amide bond (Figure 1b) [11,12]. This process, termed ‘native chemical ligation’ (NCL), has been widely applied to the synthesis of numerous moderately sized peptides and proteins [4].
Introduction The chemical synthesis of proteins has traditionally involved the sequential coupling of amino acids on a solid support. Decades of optimization have made stepwise solid-phase peptide synthesis (SPPS) a routine and reliable tool for preparing peptides up to 20–30 amino acids long [1]. In favorable cases, even longer polymers can be produced, as illustrated by the synthesis of ribonuclease A (124 amino acids) [2] and HIV protease (99 amino acids) [3]. In general, however, yields decrease with increasing length of the polypeptide and removal of the impurities that accumulate during the synthesis of longer polymers can often require heroic effort. Convergent assembly of proteins from peptide fragments largely overcomes the limitations of a purely stepwise approach and significantly extends the size of molecules that can be prepared in the laboratory [4,5].
Chemoselective ligations Convergent strategies for the synthesis of proteins depend directly on the availability of chemoselective www.current-opinion.com
The NCL strategy is general and quite robust. In addition to cysteine, many other nucleophilic residues can mediate chemical ligation, including homocysteine [13], selenocysteine [14–16], selenohomocysteine [17] and histidine [18]. An even broader range of formal ligation sites can be accessed by reducing the C–S [19] or C–Se [20] bond after the amide bond has formed, or by using removable sulfurcontaining auxiliaries to facilitate fragment condensation [21,22,23]. In some cases, it is possible to perform ligations with donor fragments lacking a nucleophilic sidechain at their N terminus, for example when the reaction is carried out under conditions favoring a folded state that holds the reactive amine proximal to the thioester [24]. When larger proteins are desired, serial ligations of multiple peptide segments can be performed, although special protecting groups or orthogonal coupling methods may be required. The Staudinger ligation, which forms an amide bond from an azide and a specifically functionalized phosphine (Figure 1b), is an example of the latter. Alkyl Current Opinion in Structural Biology 2003, 13:589–594
590 Biophysical methods
Figure 1
through SPPS alone, as shown by the semisynthesis of analogs of the b0 subunit of Escherichia coli RNA polymerase, which contains 1407 amino acid residues [30]. EPL also makes possible segmental labeling of large proteins [31], which is of considerable benefit to NMR structural studies.
(a)
+
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State of the art Peptide 2
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(b) NCL XH
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‘Traceless’ Staudinger ligation O
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Over the past year, chemoselective ligations have been exploited in many different ways. They have been used to prepare membrane proteins [32], enzymes containing unnatural amino acids for detailed investigations of chemical mechanism [33], and proteins modified with biophysical probes to explore protein folding and dynamics, and interactions between protein domains [17,34]. Also, a selectively lipidated protein was synthesized for studies of post-translational prenylation [35]. The fact that the number and nature of modifications are determined completely by the synthetic protocol, affording homogenously modified proteins, contrasts with cellular post-translational processes which often yield heterogeneous mixtures.
azides react with triarylphosphines to form aza-ylides [25]. If the aza-ylide is produced adjacent to an ester or thioester, intramolecular acyl transfer occurs efficiently to afford a new amide bond. Subsequent hydrolysis liberates the ligated peptide plus phosphine oxide. ‘Traceless’ versions of this method have been developed that leave no residual atoms from the triarylphosphine prosthetic group in the product [26–28]. The great advantage of the Staudinger ligation is that the donor fragment does not require an N-terminal residue with a nucleophilic sidechain. Thus, it can be applied in combination with NCL to generate larger proteins, as illustrated by the preparation of an isotopically labeled version of the enzyme ribonuclease A [29].
Importantly, one need not adhere to nature’s constraints, as shown by the preparation of an artificial variant of erythropoietin (Epo), a glycoprotein hormone that normally regulates the proliferation, differentiation and maturation of erythroid cells. The synthesis of this 166 amino acid protein, covalently linked to two negatively charged, branched and monodisperse synthetic polymers rather than to natural carbohydrates, is a tour de force [36]. Two peptide–polymer conjugates were initially constructed by a chemoselective oxime-forming ligation reaction between an aminoxy group on the polymer and a ketone-bearing lysine on the respective peptides; these were then coupled with two other peptide segments using thioligation methods (Figure 2). After the first two fragment condensations, the cysteine residues involved were alkylated with bromoacetic acid to provide an amino acid at the ligation sites that is electronically and sterically similar to the glutamate residue found at the corresponding positions in Epo. This procedure provided more than 100 mg of polymer-modified Epo. The artificial hormone was shown to have potent haematopoietic activity in cell and animal assays, with notably prolonged duration of action in vivo relative to natural Epo. The design and synthesis of this 51 kDa macromolecule impressively illustrates how modern chemoselective ligation strategies can provide precise control over covalent structure and molecular function. In principle, the properties of many proteins of pharmaceutical interest could be similarly fine-tuned.
In many instances, one or more of the peptide segments used to assemble a protein is produced recombinantly and the assembly process is then referred to as ‘expressed protein ligation’ (EPL) [10]. Facile production of recombinant fragments has the decided advantage that much larger molecules can be tackled than are accessible
The same ligation strategies that have proven so efficacious in constructing polypeptides can also be expected to find wide application in the construction of artificial polymers based on non-natural backbones. As a case in point, b-peptides are efficiently coupled using thioligation chemistry [37]. Chimeric proteins, in which nonpeptidic
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P R R Current Opinion in Structural Biology
Chemoselective ligations. (a) Schematic representation of peptide fragment condensation. (b) NCLs and Staudinger ligations are chemoselective and orthogonal ways of forming amide bonds between peptide segments. P1 and P2 represent the N- and C-terminal peptides, respectively.
Current Opinion in Structural Biology 2003, 13:589–594
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Figure 2
(Acm)S SR
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O 24
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(ii) Three successive ligations H N
SEP(1–32) O N O
H N
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(iii) Folding and disulfide bond formation
Current Opinion in Structural Biology
Synthesis of a synthetic erythropoiesis protein (SEP). Four unprotected peptide fragments were produced by SPPS. Two of the fragments, SEP (1–32) and SEP (117–166), which contain nonstandard Ne-levulinyl lysine residues, were subsequently modified via an oxime-forming reaction with an anionic, branched and monodisperse synthetic polymer (i). The mainchain was then assembled from the C to the N terminus by three successive NCLs (ii). After the first two condensations, the cysteine residues at positions 89 and 117 were alkylated with bromoacetic acid to mimic the glutamate residues found at the corresponding positions in Epo. The full-length peptide–polymer conjugate was folded under oxidative conditions to give the biologically active artificial hormone (iii). The peptide is shown as a ribbon diagram (blue) based on the coordinates for Epo (PDB entry 1BUY) and the branched polymers (red) are scaled to represent the approximate hydrodynamic volume of SEP [36].
elements are incorporated into the peptide backbone, have also been prepared, including a ribonuclease derivative containing a b-peptide reverse turn [38]. Ready access to other non-natural polymers of defined length and composition is likely to afford many exciting opportunities in medicine, material science and catalyst design [39].
Protein semisynthesis in vivo The burgeoning revolution in proteomics is fueling the need for proteins with tailored modifications. Designer proteins are facilitating efforts to characterize the movement, interactions and chemical microenvironment of macromolecules inside living cells [40], as well as the construction of protein microarrays for defined assays in vitro [41]. A common biological approach is to create recombinant fusion proteins, in which the protein of interest is covalently linked to another protein, such as glutathione S-transferase or green fluorescent protein (GFP) [42,43], which provide convenient handles for purification, targeting or biophysical investigation. If www.current-opinion.com
proteins contain uniquely reactive sites, they can also be selectively modified with cell-permeable fluorophores or other probes [44,45]. Alternatively, suppressor tRNAs and engineered tRNA synthetases are being developed that may eventually enable the site-selective incorporation of virtually any nonstandard amino acid into any protein in the proteome [46]. Each approach has advantages and disadvantages, and the method of choice will ultimately depend on the envisaged application. Convergent protein synthesis has considerable potential as a complementary approach for engineering proteins intracellularly. The first steps in this direction have already been taken, with trans-splicing reactions mediated by inteins playing a key role. In protein trans-splicing, the intein is split into two components. These must assemble to initiate the autocatalytic process that leads to excision of the intein fragments and ligation of the flanking N- and C-terminal domains. Such systems have already been modified to produce cyclic peptides and proteins that Current Opinion in Structural Biology 2003, 13:589–594
592 Biophysical methods
exhibit enhanced thermodynamic stability and increased resistance to exo-peptidase digestion in vivo [47–51,52]. The cyclic products can also be screened directly for antibiotic activity or other interesting properties. For example, a split intein has been engineered into a retroviral expression system for the intracellular delivery of random libraries of cyclic peptides to human cells [53]. Coupled with a functional genetic screen, this method afforded cyclic pentapeptides that selectively block interleukin-4 signaling. As shown in Figure 3, protein trans-splicing also provides a means of selectively labeling recombinant proteins inside a cell. The target molecule is biosynthesized intracellularly with one-half of the split intein (IN) fused to its C terminus. The other half of the intein (IC) is fused to the molecular probe and linked via a disulfide bond to a protein transduction domain (PTD) peptide. When this semisynthetic construct is added to the medium, the PTD peptide carries it into the cell, where the disulfide bond is cleaved, the split inteins assemble and protein Figure 3
PTD Probe
C
IC Delivery and reduction DNA PTD Expression
Protein
C
IC
IN
Probe
Complementation
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splicing occurs to link the target protein to the probe. The feasibility of this scheme has been demonstrated through semisynthesis of a GFP variant bearing a C-terminal FLAG epitope [54]. In the future, however, it should be possible to exploit trans-splicing to couple many peptides and modified cysteine derivatives to proteins in vivo. The splicing process depicted in Figure 3 is triggered by the exogenous addition of one component to cells already producing the other. Consequently, temporal control of protein modification is inherent to the system. It may also be possible to regulate protein trans-splicing inside living cells using small molecules. In fact, an artificial heterodimeric split intein has been engineered that dimerizes only upon binding of a small bidentate ligand [55]. This system has considerable promise for in vivo applications [56].
Perspectives Versatile methods for chemoselectively coupling peptides have provided a practical basis for constructing proteins in a general and modular fashion. Methodological improvements that further increase the efficiency of existing ligation chemistry, afford orthogonal ways of linking peptide segments with an extended range of permissible coupling sites, and enable synthetic manipulations in in vivo settings will make chemical synthesis of proteins an increasingly attractive alternative to purely biological production. The key advantages of the chemical approach are an exacting control over covalent structure and the ability to explore structural realms outside nature’s normal scope. In sum, chemical synthesis is a powerful enabling technology. Chemists and biologists interested in developing new materials or exploring problems in basic science — including the molecular basis of protein function and the roles proteins play in complex biochemical pathways within the cell — stand to benefit enormously from ready access to synthetic proteins with tailored properties. As we look beyond today’s horizons, toward novel nonpeptide polymeric worlds created by analogous strategies and with the same control as natural polypeptides, exciting and unexpected discoveries are sure to be made.
Acknowledgements
IN
Protein splicing
IC
Protein
C
Probe
We thank the ETH Zu¨ rich and Novartis Pharma for financial support, and Ken Woycechowsky for helpful discussions. GC is grateful to the Roche Research Foundation for a doctoral fellowship.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
Current Opinion in Structural Biology
Protein trans-splicing strategy for modifying recombinant proteins in vivo with an exogenously added probe. The two halves of the split intein are designated IC and IN; PTD is a protein transduction domain peptide that enables cellular uptake of the probe molecule. C is the cysteine involved in protein splicing and used to link the PTD to the IC probe. Current Opinion in Structural Biology 2003, 13:589–594
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22. Offer J, Boddy CNC, Dawson PE: Extending synthetic access to proteins with a removable acyl transfer auxiliary. J Am Chem Soc 2002, 124:4642-4646. An acid-labile 2-mercaptobenzyl auxiliary is described that can replace cysteine in NCLs. Its utility was demonstrated by the synthesis of a 62 amino acid SH3 domain through ligation at a Lys–Gly sequence. 23. Botti P, Carrasco MR, Kent SBH: Native chemical ligation using removable Na-(1-phenyl-2- mercaptoethyl) auxiliaries. Tetrahedron Lett 2001, 42:1831-1833. 24. Beligere GS, Dawson PE: Conformationally assisted protein ligation using C-terminal thioester peptides. J Am Chem Soc 1999, 121:6332-6333. 25. Staudinger H, Meyer J: New organic compounds of phosphorous. III. Phosphinemethylene derivatives and phosphinimines. Helv Chim Acta 1919, 2:635-646. 26. Nilsson BL, Kiessling LL, Raines RT: Staudinger ligation: a peptide from a thioester and azide. Org Lett 2000, 2:1939-1941. 27. Saxon E, Armstrong JI, Bertozzi CR: A ‘traceless’ Staudinger ligation for the chemoselective synthesis of amide bonds. Org Lett 2000, 2:2141-2143. 28. Nilsson BL, Kiessling LL, Raines RT: High-yielding Staudinger ligation of a phosphinothioester and azide to form a peptide. Org Lett 2001, 3:9-12. 29. Nilsson BL, Hondal RJ, Soellner MB, Raines RT: Protein assembly by orthogonal chemical ligation methods. J Am Chem Soc 2003, 125:5268-5269. This article describes the synthesis of ribonuclease A from three fragments, using sequential Staudinger and native chemical ligations. 30. Mukhopadhyay J, Kapanidis AN, Mekler V, Kortkhonjia E, Ebright YW, Ebright RH: Translocation of r70 with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA. Cell 2001, 106:453-463. 31. Xu R, Ayers B, Cowburn D, Muir TW: Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies. Proc Natl Acad Sci USA 1999, 96:388-393. 32. Valiyaveetil FI, MacKinnon R, Muir TW: Semisynthesis and folding of the potassium channel KcsA. J Am Chem Soc 2002, 124:9113-9120. EPL was successfully exploited to synthesize a truncated version of the KcsA potassium channel, which was subsequently incorporated into lipid vesicles. This study highlights the potential utility of ligation strategies for the (semi)synthesis of integral membrane proteins. 33. Kienho¨ fer A, Kast P, Hilvert D: Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donor. J Am Chem Soc 2003, 125:3206-3207. EPL was used to prepare a chorismate mutase variant in which an active site arginine was replaced by an isosteric but neutral citrulline. The resulting dramatic loss of catalytic activity, accompanied by relatively modest effects on substrate and inhibitor binding, provides evidence for selective electrostatic stabilization of the pericyclic transition state. 34. Camarero JA, Shekhtman A, Campbell EA, Chlenov M, Gruber TM, Bryant DA, Darst SA, Cowburn D, Muir TW: Autoregulation of a bacterial r factor explored by using segmental isotopic labeling and NMR. Proc Natl Acad Sci USA 2002, 99:8536-8541. NMR spectroscopic studies of a bacterial s factor, which was prepared in segmentally labeled form by EPL, argue against a high-affinity intramolecular interaction between two regions of this protein, previously believed to be responsible for autoinhibition of its DNA-binding function. 35. Alexandrov K, Heinemann I, Durek T, Sidorovitch V, Goody RS, Waldmann H: Intein-mediated synthesis of geranylgeranylated Rab7 protein in vitro. J Am Chem Soc 2002, 124:5648-5649. A combination of organic synthesis, protein expression and in vitro protein ligation afforded a fluorescent, monoprenylated Rab7 protein for mechanistic studies of the prenylation reaction catalyzed by Rab geranylgeranyltransferase. 36. Kochendoerfer GG, Chen SY, Mao F, Cressman S, Traviglia S, Shao H, Hunter CL, Low DW, Cagle EN, Carnevali M et al.: Design and chemical synthesis of a homogeneous polymer-modified erythropoiesis protein. Science 2003, 299:884-887. The design and total synthesis of ‘synthetic erythropoiesis protein’, a 51 kDa protein–polymer construct, in homogeneous form illustrates the Current Opinion in Structural Biology 2003, 13:589–594
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48. Evans TC, Martin D, Kolly R, Panne D, Sun L, Ghosh I, Chen L, Benner J, Liu XQ, Xu MQ: Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803. J Biol Chem 2000, 275:9091-9094. 49. Camarero JA, Fushman D, Cowburn D, Muir TW: Peptide chemical ligation inside living cells: in vivo generation of a circular protein domain. Bioorg Med Chem 2001, 9:2479-2484. 50. Iwai H, Lingel A, Plu¨ ckthun A: Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J Biol Chem 2001, 276:16548-16554. 51. Scott CP, Abel-Santos E, Jones AD, Benkovic SJ: Structural requirements for the biosynthesis of backbone cyclic peptide libraries. Chem Biol 2001, 8:801-815. 52. Williams NK, Prosselkov P, Liepinsh E, Line I, Sharipo A, Littler DR, Curmi PMG, Otting G, Dixon NE: In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein. J Biol Chem 2002, 277:7790-7798. A synthetic split mini-intein gene was constructed for intracellular cyclization of recombinant proteins produced in E. coli. It was shown to cyclize the N-terminal domain of E. coli DnaB efficiently, without accumulating significant amounts of unprocessed precursor or linear protein, as has been observed with other split intein systems. 53. Kinsella TM, Ohashi CT, Harder AG, Yam GC, Li W, Peelle B, Pali ES, Bennett MK, Molineaux SM, Anderson DA et al.: Retrovirally delivered random cyclic peptide libraries yield inhibitors of interleukin-4 signaling in human B cells. J Biol Chem 2002, 277:37512-37518. A split intein was engineered into a retroviral expression system to facilitate intracellular delivery of a library of random cyclic peptides into human cells. Cyclization of peptides was verified in cell lysates by mass spectrometry and peptides that selectively inhibit germ line epsilon transcription were identified through a functional screen. 54. Giriat I, Muir TW: Protein semi-synthesis in living cells. J Am Chem Soc 2003, 125:7180-7181. A general strategy is presented for exploiting split intein technology to modify proteins with probe molecules in vivo. Its feasibility is demonstrated by attachment of a C-terminal FLAG epitope, introduced exogenously to the growth medium and carried into the cell by a PTD peptide, to intracellular GFP. The method has broad potential for modifying proteins in their natural setting. 55. Mootz HD, Muir TW: Protein splicing triggered by a small molecule. J Am Chem Soc 2002, 124:9044-9045. An approach for regulating protein splicing is outlined. The ability of a small molecule, rapamycin, to heterodimerize proteins FKBP and FRB is exploited to bring together each half of a weakly interacting split intein fused to these two proteins. Protein splicing of the flanking sequences was shown to occur only in the presence of rapamycin. 56. Mootz HD, Blum ES, Tyszkiewicz AB, Muir TW: Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo. J Am Chem Soc 2003, 125:10561-10569. The protein splicing activity of an engineered split intein in mammalian cells was shown to be inducible by exogenous addition of the small molecule rapamycin. Response time, dose dependency and effects of inhibitors were investigated.
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