Access to site-specific Fc-cRGD peptide conjugates through streamlined expressed protein ligation.

An ideal drug should be highly effective, non-toxic and be delivered by a convenient and painless single dose. We are still far from such optimal treatment but peptides, with their high target selectivity and low toxicity profiles, provide a very attractive platform from which to strive towards it. One of the major limitations of peptide drugs is their high clearance rates, which limit dosage regimen options. Conjugation to antibody Fc domains is a viable strategy to improve peptide stability by increasing their hydrodynamic radius and hijacking the Fc recycling pathway. We report the use of a split-intein based semi-synthetic approach to site-specifically conjugate a synthetic integrin binding peptide to an Fc domain. The strategy described here allows conjugating synthetic peptides to Fc domains, which is not possible via genetic methods, fully maintaining the ability of both the Fc domain and the bioactive peptide to interact with their binding partners.

Vascular injury during tension-free vaginal tape procedure for stress urinary incontinence.

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.

Fluorescent monitoring of kinase activity in real time: development of a robust fluorescence-based assay for Abl tyrosine kinase activity.

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.

The TGF beta receptor activation process: an inhibitor- to substrate-binding switch.

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.

Peptide chemical ligation inside living cells: in vivo generation of a circular protein domain.

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.

Rescuing a destabilized protein fold through backbone cyclization.

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.

Mapping the molecular interface between the sigma(70) subunit of E. coli RNA polymerase and T4 AsiA.

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.

Native chemical ligation of polypeptides.

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.

Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling.

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.

Protein engineering by expressed protein ligation.

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.

Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC.

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.

Exfoliatin-producing strains define a fourth agr specificity group in Staphylococcus aureus.

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.

Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation.

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.

Peptide ligation and its application to protein engineering.

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.

Structure-activity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence.

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.

Virulence gene regulation by peptides in staphylococci and other Gram-positive bacteria.

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.

Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies.

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.

Introduction of unnatural amino acids into proteins using expressed protein ligation.

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.