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Expressed protein ligation (EPL) is a protein engineering approach that allows recombinant and synthetic polypeptides to be chemoselectively and regioselectively joined together. The approach makes the primary structure of most proteins accessible to the tools of synthetic organic chemistry, enabling the covalent structure of proteins to be modified in an unprecedented fashion. The ability to incorporate noncoded amino acids, biophysical probes, and stable isotopes into specific locations within proteins provides research tools to peer into the inner workings of these molecules. In this review I discuss the development of this technology, its broad application to biological systems, and its possible role in the area of proteomics.

The use of small molecules that turn specific proteins on or off provides a level of temporal control that is difficult to achieve using standard genetic approaches. Consequently, the development of small-molecule switches of protein function is a very active area of chemical biology, sometimes referred to as chemical genetics. Most studies in this area rely on the identification of small molecules that bind directly to the active site of a target protein, thereby acting as agonists or antagonists of function. Strategies have also been described in which the small molecule triggers a change in the secondary, tertiary, or ternary structure of the protein, in so doing changing the functional state of the molecule. Another approach to this problem would be to alter the primary structure of a target protein in response to a small-molecule trigger; a dramatic change in primary sequence would be directly coupled to function. In principle, this can be achieved by harnessing protein splicing, a posttranslational editing process that results in the precise removal of an internal domain (termed an intein) from two flanking sequences termed the N- and C-exteins. In this communication we introduce a technique that allows protein splicing to occur only in the presence of the small molecule, rapamycin. This approach is expected to be independent of the nature of the two exteins and so should provide a general vehicle for controlling protein function using small molecules.

In this contribution we describe the semisynthesis of the potassium channel, KcsA. A truncated form of KcsA, comprising the first 125 amino acids of the 160-amino acid protein, was synthesized using expressed protein ligation. This truncated form corresponds to the entire membrane-spanning region of the protein and is similar to the construct previously used in crystallographic studies on the KcsA protein. The ligation reaction was carried out using an N-terminal recombinant peptide alpha-thioester, corresponding to residues 1-73 of KcsA, and a synthetic C-terminal peptide corresponding to residues 74-125. Chemical synthesis of the C-peptide was accomplished by optimized Boc-SPPS techniques. A dual fusion strategy, involving glutathione-S-transferase (GST) and the GyrA intein, was developed for recombinant expression of the N-peptide alpha-thioester. The fusion protein, expressed in the insoluble form as inclusion bodies, was refolded and then cleaved successively to remove the GST tag and the intein, thereby releasing the N-peptide alpha-thioester. Following chemical ligation, the KcsA polypeptide was folded into the tetrameric state by incorporation into lipid vesicles. The correctness of the folded state was verified by the ability of the KcsA tetramer to bind to agitoxin-2. To our knowledge, this work represents the first reported semisynthesis of a polytopic membrane protein and highlights the potential application of native chemical ligation and expressed protein ligation for the (semi)synthesis of integral membrane proteins.

Staphylococcal pathogenesis is regulated by a two-component quorum-sensing system, agr, activated upon binding of a self-coded autoinducing peptide (AIP) to the receptor-histidine kinase, AgrC. The AIPs consist of a thiolactone macrocyle and an exocyclic "tail", both of which are important for function. In this report, characterization of the unique AIPs from the four known agr specificity groups of Staphylococcus aureus has been completed, along with analysis of cross-group inhibition of AgrC activation by each of the four AIPs. The following conclusions have been drawn: (i) The native thiolactone macrocyle and tail are necessary and sufficient for full activation by the AIPs, whereas the AIP-I macrocycle alone is a partial agonist. (ii) The native N-terminus is less critical, as that of AIP-I can be modified without affecting bioactivity, although that of AIP-III cannot. (iii) The ring and tail may function differently in different AIPs. Thus the group I and IV AIPs differ at a single (endocyclic) residue, which is the determinant of AIP specificity for these two groups and is essential for function. A similarly critical residue in AIP-II, however, is exocyclic. (iv) Cross-inhibition is more tolerant of sequence and structural diversity than is activation, suggesting that the AIPs interact differently with cognate than with heterologous receptors. (v) Chimeric peptides, in which the tails and macrocycles are switched, do not activate and instead inhibit receptor activation. These data suggest a model in which activation and inhibition involves different binding orientations within the ligand binding pocket of each receptor.

Expressed protein ligation is a technique for joining recombinantly expressed proteins to polypeptides containing biophysical probes, post-translational modifications or unnatural amino acids. Recent advances have expanded the scope of expressed protein ligation and have allowed the approach to be applied to the study of basic biological questions.

Bacterial sigma factors combine with the catalytic core RNA polymerase to direct the process of transcription initiation through sequence-specific interactions with the -10 and -35 elements of promoter DNA. In the absence of core RNA polymerase, the DNA-binding function of sigma is autoinhibited by its own N-terminal 90 amino acids (region 1.1), putatively by a direct interaction with conserved region 4.2, which binds the -35 promoter element. In the present work, this mechanism of autoinhibition was studied by using a combination of NMR spectroscopy and segmental isotopic labeling of a sigma70-like subunit from Thermotoga maritima. Our data argue strongly against a high-affinity interaction between these two domains. Instead we suggest that autoinhibition of DNA binding occurs through an indirect steric and/or electrostatic mechanism. More generally, the present work illustrates the power of segmental isotopic labeling for probing molecular interactions in large proteins by NMR.

Staphylococcal pathogenesis is regulated by a two-component quorum-sensing system, agr, activated by a self-coded autoinducing peptide (AIP). The agr system is widely divergent and is unique in that variant AIPs cross-inhibit agr activation in heterologous combinations. Cross-inhibition, but not self-activation, is widely tolerant of structural diversity in the AIPs so that these two processes must involve different mechanisms of interaction with the respective receptors. Herein, we have utilized this naturally occurring antagonism to demonstrate that both activation and inhibition are reversible and that activators and inhibitors interact at a common site on the receptor. These results suggest that molecules designed to compete with natural agonists for binding at receptor-histidine kinase sensor domains could represent a general approach to the inhibition of receptor-histidine kinase signaling.

[structure: see text] Semisynthesis of an active tetraphosphorylated analogue of the Type I TGFbeta receptor is reported. An efficient native chemical ligation protocol was developed to link a tetraphosphopeptide and a recombinant receptor fragment. Synthesis of the peptide alpha-thioester on a 4-sulfamylbutyryl resin was optimized following the characterization of a major side reaction and subsequent substitution of norleucine for methionine in the peptide sequence. These optimized protocols will be applicable to the semisynthesis of related protein kinases.

Fluorescent biosensors hold great promise for drug discovery. Using a solid-phase version of protein semi-synthesis, we incorporated two fluorophores at specific sites within a truncated version of the c-Crk-II protein. The resulting fluorescent protein biosensor permits the real-time monitoring of Abl kinase activity and provides a robust and rapid method for assaying Abl kinase inhibitors.

Ligand-induced phosphorylation of the receptor-regulated Smads (R-Smads) is essential in the receptor Ser/Thr kinase-mediated TGF-beta signaling. The crystal structure of a phosphorylated Smad2, at 1.8 A resolution, reveals the formation of a homotrimer mediated by the C-terminal phosphoserine (pSer) residues. The pSer binding surface on the MH2 domain, frequently targeted for inactivation in cancers, is highly conserved among the Co- and R-Smads. This finding, together with mutagenesis data, pinpoints a functional interface between Smad2 and Smad4. In addition, the pSer binding surface on the MH2 domain coincides with the surface on R-Smads that is required for docking interactions with the serine-phosphorylated receptor kinases. These observations define a bifunctional role for the MH2 domain as a pSer-X-pSer binding module in receptor Ser/Thr kinase signaling pathways.

Background: Tension-free vaginal tape procedure is a popular surgical treatment of genuine stress urinary incontinence., Cases: Two cases of retropubic hematoma after tension-free vaginal tape procedure are reported. One woman with an 8 x 10 cm hematoma localized to the retropubic space required transfusion of two units of packed red blood cells for symptomatic relief. Neither case required reoperation, and both patients' hematomas resolved over 6 months without treatment. Both patients were continent 9-12 months after surgery., Conclusion: Although the tension-free vaginal tape procedure is a minimally invasive operation for stress urinary incontinence and appears to be effective, significant vascular complications can result.

Here we describe the first example of a peptide chemical ligation reaction performed inside a living cell. A cell-based native chemical ligation approach was developed and used to generate a circular version of the N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein inside Escherichia coli cells. The in vivo cyclization reaction was extremely efficient and the resulting circular protein domain was fully biologically active and able to adopt the native SH3 folded structure. This work represents an important step towards the in vivo generation of small backbone cyclic peptides for use in basic biological research.

The type I TGF beta receptor (T beta R-I) is activated by phosphorylation of the GS region, a conserved juxtamembrane segment located just N-terminal to the kinase domain. We have studied the molecular mechanism of receptor activation using a homogeneously tetraphosphorylated form of T beta R-I, prepared using protein semisynthesis. Phosphorylation of the GS region dramatically enhances the specificity of T beta R-I for the critical C-terminal serines of Smad2. In addition, tetraphosphorylated T beta R-I is bound specifically by Smad2 in a phosphorylation-dependent manner and is no longer recognized by the inhibitory protein FKBP12. Thus, phosphorylation activates T beta R-I by switching the GS region from a binding site for an inhibitor into a binding surface for substrate. Our observations suggest that phosphoserine/phosphothreonine-dependent localization is a key feature of the T beta R-I/Smad activation process.

We describe the physicochemical characterization of various circular and linear forms of the approximately 60 residue N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein. Structural, dynamic, thermodynamic, kinetic and biochemical studies reveal that backbone circularization does not prevent the adoption of the natural folded structure in any of the circular proteins. Both the folding and unfolding rate of the protein increased slightly upon circularization. Circularization did not lead to a significant thermodynamic stabilization of the full-length protein, suggesting that destabilizing enthalpic effects (e.g. strain) negate the expected favorable entropic contribution to overall stability. In contrast, we find circularization results in a dramatic stabilization of a truncated version of the SH3 domain lacking a key glutamate residue. The ability to rescue the destabilized mutant indicates that circularization may be a useful tool in protein engineering programs geared towards generating minimized proteins., (Copyright 2001 Academic Press.)

The total synthesis and semisynthesis of proteins allows the site-specific incorporation of unnatural amino acids, post-translational modifications, and biophysical/biochemical probes into the target molecule. Among the various chemical and enzymatic approaches available for the synthesis/semisynthesis of proteins, the native chemical ligation technique has proven especially useful and is the exclusive focus of this unit. This unit first discusses how to choose the ligation site(s) in the target protein and then outlines how to obtain the necessary polypeptide building blocks using either chemical synthesis or recombinant DNA expression. Next, the synthesis of a protein by native chemical ligation of two polypeptide fragments is described. The synthesis of a protein from three polypeptide fragments using a sequential native chemical ligation strategy is also described. Support protocols describe how to obtain the necessary polypeptide fragments using either chemical synthesis or recombinant DNA expression.

Bacteriophage T4 antisigma protein AsiA (10 kDa) orchestrates a switch from the host and early viral transcription to middle viral transcription by binding to the sigma(70) subunit of E. coli RNA polymerase. The molecular determinants of sigma(70)-AsiA complex formation are not known. Here, we used combinatorial peptide chemistry, protein-protein crosslinking, and mutational analysis to study the interaction between AsiA and its target, the 33 amino acid residues-long sigma(70) peptide containing conserved region 4.2. Many region 4.2 amino acid residues contact AsiA, which likely completely occludes the DNA-binding surface of region 4.2. Though none of region 4.2 amino acid residues is singularly responsible for the very tight interaction with AsiA, sigma(70) Lys593 and Arg596 which lie outside the putative DNA recognition element of region 4.2, contribute the most. In AsiA, the first 20 amino acid residues are both necessary and sufficient for interactions with sigma(70). Our results clarify details of sigma(70)-AsiA interaction and open the way for engineering AsiA derivatives with altered specificities., (Copyright 2001 Academic Press.)

Two-component signaling systems involving receptor-histidine kinases are ubiquitous in bacteria and have been found in yeast and plants. These systems provide the major means by which bacteria communicate with each other and the outside world. Remarkably, very little is known concerning the extracellular ligands that presumably bind to receptor-histidine kinases to initiate signaling. The two-component agr signaling circuit in Staphylococcus aureus is one system where the ligands are known in chemical detail, thus opening the door for detailed structure-activity relationship studies. These ligands are short (8- to 9-aa) peptides containing a thiolactone structure, in which the alpha-carboxyl group of the C-terminal amino acid is linked to the sulfhydryl group of a cysteine, which is always the fifth amino acid from the C terminus of the peptide. One unique aspect of the agr system is that peptides that activate virulence expression in one group of S. aureus strains also inhibit virulence expression in other groups of S. aureus strains. Herein, it is demonstrated by switching the receptor-histidine kinase, AgrC, between strains of different agr specificity types, that intragroup activation and intergroup inhibition are both mediated by the same group-specific receptors. These results have facilitated the development of a global inhibitor of virulence in S. aureus, which consists of a truncated version of one of the naturally occurring thiolactone peptides.

The staphylococcal virulon is activated by the density-sensing agr system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by agrD. A central segment of the agr locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of Staphylococcus aureus isolates into three different agr specificity groups and to the division of non-aureus staphylococci into a number of others. The AIPs cross-inhibit the agr responses between groups. We have previously shown that most menstrual toxic shock strains belong to agr specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth agr specificity group among S. aureus strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the agr response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the agr autoinduction threshold.

Background: The site-specific chemical modification of proteins has proved to be extremely powerful for generating tools for the investigation of biological processes. Although a few elegant methods exist for engineering a recombinant protein at a unique position, these techniques cannot be easily extended to allow several different chemical probes to be specifically introduced into a target sequence. As such multiply labeled proteins could be used to study many biological processes, and in particular biomolecular interactions, we decided to investigate whether such protein reagents could be generated using an extension of the semisynthesis technique known as expressed protein ligation., Results: A solid-phase expressed protein ligation (SPPL) technology is described that enables large semisynthetic proteins to be assembled on a solid support by the controlled sequential ligation of a series of recombinant and synthetic polypeptide building blocks. This modular approach allows multiple, different chemical modifications to be introduced site-specifically into a target protein. This process, which is analogous to solid-phase peptide synthesis, was used to dual-label the amino and carboxyl termini of the Crk-II adapter protein with the fluorescence resonance energy transfer pair tetramethylrhodamine and fluorescein, respectively. The resulting construct reports (through a fluorescence change) the phosphorylation of Crk-II by the nonreceptor protein tyrosine kinase, c-Abl, and was used to probe the protein-protein interactions that regulate this important post-translational process., Conclusions: SPPL provides a powerful method for specifically modifying proteins at multiple sites, as was demonstrated by generating a protein-based biosensor for Crk-II phosphorylation. Such protein derivatives are extremely useful for investigating protein function in vitro and potentially in vivo. This modular approach should be applicable to many different protein systems.

By allowing the controlled assembly of synthetic peptides and recombinant polypeptides, expressed protein ligation permits unnatural amino acids, biochemical probes, and biophysical probes to be specifically incorporated into semisynthetic proteins. A powerful feature of the method is its modularity; once the reactive recombinant pieces are in hand and the optimal ligation conditions have been developed, it is possible to quickly generate an array of semisynthetic analogs by simply attaching different synthetic peptide cassettes--in most cases the synthetic peptides will be small and easy to make. From a practical perspective, the rate-determining step in the process is usually not the ligation step (it is based on a simple and efficient chemical reaction), but rather the generation of the reactive polypeptide building blocks. In particular, optimizing the yields of recombinant polypeptide building blocks can require some initial effort. However, it should be noted that the initial investment in time required to optimize the production of the recombinant fragment is offset by the ease and speed with which one can produce the material thereafter. In the example described in this chapter, the yield of soluble intein fusion protein was slightly better using the GyrA intein than for the VMA intein, although in both cases significant amounts of fusion protein were present in the cell pellet. Studies are currently underway to identify optimal refolding conditions for GyrA fusion proteins solubilized from inclusion bodies.

The ability to assemble a target protein from a series of peptide fragments, either synthetic or biosynthetic in origin, enables the covalent structure of a protein to be modified in an unprecedented fashion. The present technologies available for performing such peptide ligations are discussed, with an emphasis on how these methodologies have been utilized in protein engineering to investigate biological processes.

The synthesis of virulence factors and other extracellular proteins responsible for pathogenicity in Staphylococcus aureus is under the control of the agr locus. A secreted agr-encoded peptide, AgrD, processed from the AgrD gene product, is known to be an effector of self-strain activation and cross-strain inhibition of the agr response. Biochemical analysis of AgrD peptides isolated from culture supernatants has suggested that they contain an unusual thiol ester-linked cyclic structure. In the present work, chemical synthesis is used to confirm that the mature AgrD peptides contain a thiolactone structure and that this feature is absolutely necessary for full biological activity. The AgrD synthetic thiolactone peptides exhibited biological activity in vivo in a mouse protection test. Structure-activity studies have allowed key aspects of the peptide structure involved in the differential activation and inhibition functions to be identified. Accordingly, we propose a model for activation and inhibition of the agr response in which the former, but not the latter, involves specific acylation of the agr transmembrane receptor, AgrC.

In staphylococci, autoinducing peptides activate agr. a global regulator of the expression of genes encoding virulence factors and other exoproteins. During the past year, there have been major advances in the structure-function analysis of these peptides and the regulation of a virulence factor by an autoinducing peptide in pneumococci has been demonstrated.

A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins., (Copyright 2000 John Wiley & Sons, Inc.)

Expressed protein ligation is a novel protein semi-synthesis method that permits the in vitro ligation of a chemically synthesized C-terminal segment of a protein to a recombinant N-terminal segment fused through its C terminus to an intein protein splicing element. In principle, the practical convenience of this method, combined with the expanded opportunities in protein engineering that it provides, makes it well suited for probing the molecular basis of complex processes such as transcription. Here we describe the successful application of expressed protein ligation to the approximately 600 amino acid sigma70 subunit of Escherichia coli RNA polymerase. The resulting semi-synthetic sigma70 constructs are shown to be fully functional and have been used to map the binding region of the bacteriophage T4 anti-sigma protein, AsiA, to within amino acids 567-600 of sigma70. The success of these semi-synthesis studies sets the stage for the future generation of semi-synthetic sigma70 molecules in which unnatural amino acids and biophysical probes are site-specifically incorporated in the RNA polymerase complex.

A protein semisynthesis method-expressed protein ligation-is described that involves the chemoselective addition of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size.

A novel technique is described that allows the synthesis, functional analysis, and quantitative readout of defined arrays of polypeptide analogues in aqueous solution. Key to this approach is the use of a simple encoding-decoding system in which a unique Fmoc-amino acid tag is covalently attached to the C terminus of each member of a molecular array through a selectively cleavable bond. These tags can be cleanly removed from the molecules they encode, allowing single-step characterization and quantification of the entire mixture by HPLC. The utility of this technique is illustrated through the preparation of an array of proline-rich sequences based on the exchange factor C3G, one of the natural ligands of the N-terminal SH3 domain from the proto-oncogene, c-Crk. The array was designed to systematically modify those residues within the C3G peptide ligand thought to make key interactions with the c-Crk SH3 domain. Using competition binding experiments, it was possible to determine the relative ED50 values for the entire array of molecules simultaneously. These studies revealed that in order to maintain optimal binding to the SH3 domain, the P-3 side chain of the ligand must be positively charged and the P-0 side chain must be hydrophobic and extend beyond the gamma-carbon. The excellent correlation between these relative ED50 values and a series of relative Kd values determined from individual peptides suggests that this approach may be useful in determining, in a parallel fashion, the relative biological activities of arrays of polypeptides.

In this article we describe a new, convenient procedure to carry out intramolecular (cyclization) and intermolecular native chemical ligations of unprotected peptides directly from a solid support. Our solid-phase ligation approach eliminates the need to manipulate peptide (alpha)thioacid and peptide (alpha)thioester intermediates in aqueous solution before the ligation step, thereby leading to a reduction in handling losses and significantly increasing the overall efficiency of the chemical ligation strategy. A key step in our ligation scheme is the ability to generate fully unprotected peptides tethered to a solid support through an (alpha)thioester linkage. This can be achieved efficiently using optimized Boc-solid-phase peptide synthesis on a 3-mercaptopropionamide-polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer support (HS-PEGA). Once the synthesis is complete, the fully protected peptide (alpha)thioester resin is treated with HF to give the corresponding fully unprotected peptide (alpha)thioester resin. Using this procedure several polypeptides ranging from 15 to 47 residues were synthesized successfully. These peptide-resins were then used to perform both intramolecular (head-to-tail cyclizations) and intermolecular solid-phase ligations. The intramolecular solid-phase ligations proceeded much faster than their intermolecular counterparts, but in both cases the reactions were observed to be remarkably clean. The presence of aromatic thiol cofactors significantly accelerated the relatively slow intermolecular ligations. This novel methodology was then extended to provide a general method for performing sequential intermolecular ligations, allowing easy access to much larger polypeptide and protein systems.

Extremely fast cyclization of the linear polypeptide precursor 1 takes place to form 2. The reaction appears to be assisted by the native fold of 1, which positions the reactive ends in close proximity. The circular topology has no influence on the folding or function of 2., (© 1998 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)

Ubiquitin is a 76-amino acid protein involved in the targeting for destruction of proteins in the cell. The protein can readily be synthesized chemically affording an extra dimension to studies of protein stability. Ubiquitin with various modifications to the hydrophobic core has been synthesized. In particular, two core amino acids have been replaced by aminobutyric acid (Val-26) and norvaline (for Ile-30) and the product crystallized. The refined crystal structure shows an overall contraction of the molecule and the side chain of Nva-30 rotates relative to Ile-30. However, the side chain rotation is not sufficient to compensate for the effect of the loss of the methyl group and hence a small cavity is introduced into the structure, which decreases the stability of the protein. The biological behaviour of the modified protein is unaltered. The observed changes in stability are of the magnitude expected for the removal of methyl groups from the hydrophobic core of a protein. Interestingly, the effect appears to be independent of the position of the removed methyl group. The intact structure, but not its stability, is important for recognition by the biological conjugating system.

Background: Modifying the covalent structure of a protein is an effective empirical route to probing three-dimensional structure and biological function. Here we describe a combinatorial protein chemistry strategy for studying structure-activity relationships in proteins. Our approach (termed 'protein signature analysis') involves functional selection from an array of self-encoded protein analogs prepared by total synthesis, coupled to a simple chemical readout that unambiguously identifies the modified proteins in the resulting active and inactive populations., Results: Protein signature analysis was used to study the interaction of the amino-terminal SH3 domain from the cellular adaptor protein c-Crk with its cognate proline-rich peptide, C3G. Using a functional selection assay, the qualitative effects of scanning a series of synthetic analog units through the amino-acid sequence of the SH3 domain were evaluated. The analog units were designed to alter both amino-acid sidechains and the polypeptide backbone within the protein. These chemical studies revealed that the sidechain of Asp 150 in the SH3 domain is essential for ligand binding and that changes in the structure of the polypeptide backbone can also result in loss of binding activity., Conclusions: These chemical studies have provided new insight into how ligand binding is related to the covalent structure of the SH3 domain. Protein signature analysis is a powerful and conceptually novel way of studying the molecular and chemical basis of protein function; it combines the advantages of systematic modification of a protein's chemical structure with the practical convenience of combinatorial synthesis.

A precise and rapid method for identifying sites of interaction between proteins was demonstrated; the basis of the method is direct mass spectrometric readout from the complex to determine the specific components of the proteins that interact--a method termed affinity-directed mass spectrometry. The strategy was used to define the region of interaction of a protein growth factor with a monoclonal antibody. A combination of proteolytic digestion and affinity-directed mass spectrometry was used to rapidly determine the approximate location of a continuous binding epitope within the growth factor. The precise boundaries of the binding epitope were determined by affinity-directed mass spectrometric analysis of sets of synthetic peptide ladders that span the approximate binding region. In addition to the mapping of such linear epitopes, affinity-directed mass spectrometry can be applied to the mapping of other types of molecule-molecule contacts, including ligand-receptor and protein-oligonucleotide interactions.

Electrospray mass spectrometry (ESMS) has been used to investigate the structural properties of a protein prepared by total chemical synthesis. Construction of an analog of the tenth type III module from fibronectin (10F3) by chemical ligation of the unprotected synthetic peptides 10F3(1-40) alpha COSH and BrAc (42-94) 10F3 was found to give two major products, both of which possessed a mass corresponding to the expected product, [(COS)40-41]10F3. Comparisons of the ESMS charge distributions obtained for these two synthetic products with that obtained for recombinant 10F3 suggested that one of the synthetic 10F3 analogs was correctly folded and the other was somehow misfolded. This was further confirmed by 1D and 2D NMR analysis. Exposure of the misfolded synthetic [(COS)40-41]10F3 to high pH and elevated temperature followed by analysis using liquid chromatography-mass spectrometry revealed a beta-ester linkage between residues Asn42 and Ser43, produced by an N-->O acyl shift rearrangement at Ser43, as the origin of the misfolding. ESMS was also used to measure the H-D exchange rates of labile protons within the synthetic and recombinant 10F3s. This application, which allows the number of slow exchanging backbone amides within a protein to be calculated, revealed clear differences in the H-bonding networks of the folded and unfolded synthetic protein modules. Replacement of Ser43 by an alanine was found to circumvent the N-->O acyl shift, and the resulting synthetic protein analogue, [Ala43, (COS)40-41]10F3, possessed identical structural properties to recombinant 10F3.

A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chemical ligation was demonstrated by the one-step preparation of a cytokine containing multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-containing protein molecule. Native chemical ligation is an important step toward the general application of chemistry to proteins.

Integrins are a class of heterodimeric cell adhesion receptors involved in cell migration, cell anchorage, and cell-cell interactions. The cytoplasmic domains of integrins are of key importance in these activities. We have designed and chemically synthesized a 126 amino acid model protein (MP-1) containing both cytoplasmic tails of the platelet-derived integrin alpha IIb beta 3 covalently linked via a helical coiled coil. The coiled-coil tertiary structure was incorporated to mimic the membrane-spanning domain of the integrin and to act as a topological constraint fixing the two cytoplasmic tails in a parallel arrangement. This molecule, which contains two C-termini, was constructed by chemical dovetailing. The bromoacetylated and cysteinyl peptide synthons were unambiguously ligated through the formation of a thioether linkage. Ultraviolet circular dichroism (CD) spectroscopy has been performed on MP-1 and related compounds, confirming that a helical coiled coil is present within the MP-1 molecule. Significantly, the helicity apparently extends beyond the predicted amphiphilic region of MP-1. Fluorescence measurements suggest that a defined tertiary structure has formed by the association of the two cytoplasmic domains. We conclude that this is a practical design strategy for the study of the cytoplasmic domain of multisubunit cell-surface receptors.

The small protein ubiquitin (76 amino acids) has been synthesized under optimized conditions by Merrifield solid-phase methodology using the N alpha-Fmoc protecting group. The crude polypeptide mixture was purified to homogeneity by gel filtration, dialysis and a combination of cation- and anion-exchange chromatography to yield ubiquitin. Amino acid analysis, enzymic digestion and sequencing by automated Edman degradation were used to authenticate the primary structure. Isoelectric focusing and m.s. were used to demonstrate that the final product was greater than 98% pure with a final yield of 93 mg (4.3%) from a single synthesis on a 0.25 nmol scale.

The solid-phase chemical synthesis of ubiquitin produced a molecule with physicochemical properties similar to those of the natural protein. We have grown crystals of this synthetic ubiquitin and performed an X-ray analysis at 1.8 A resolution in order to compare the synthetic protein with the known natural structure. The crystals were isomorphous with those of the natural protein, the R-factor between them being 7.1%. Difference Fourier analysis shows that the synthetic and natural structures are indistinguishable. The co-ordinates of the natural ubiquitin (1UBQ) were used as the starting point for restrained least-squares refinement (TNT program) against the synthetic X-ray data. The refinement converged to R = 16.5% and the resulting model did not change when refined against natural ubiquitin X-ray data (R = 18.7%). From both the refinement and featureless difference Fourier synthesis, we conclude that the synthetic and natural protein structures are identical. A short discussion about the uses of proteins with 'non-standard' amino acid residues is included.

Total chemical synthesis is developing as an important approach to the construction of native proteins and their analogs for the study of the structural basis of biochemical activity. In the past year, a radical departure from conventional synthetic approaches has led to a renaissance in this field. The inherent simplicity of these new modes of protein construction will allow large synthetic proteins (> 200 amino acids in length) to be routinely assembled; this will vastly expand the repertoire of protein molecules accessible to research.