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Biomedical subjects

T W Muir

Publications and source records attributed to T W Muir.

At least 19 recordsLinked to original sources
PubMed1999

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.

Abscess↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1998

Expressed protein ligation, a novel method for studying protein-protein interactions in transcription.

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.

Bacterial Proteins↗
PubMed1998

Expressed protein ligation: a general method for protein engineering.

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.

Animals↗
PubMed1998

Studying receptor-ligand interactions using encoded amino acid scanning.

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.

Amino Acids↗
PubMed1998

Chemical ligation of unprotected peptides directly from a solid support.

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.

Amino Acid Sequence↗
PubMed1997

Synthetic, structural and biological studies of the ubiquitin system: synthesis and crystal structure of an analogue containing unnatural amino acids.

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.

Aminobutyrates↗
PubMed1996

Mapping protein-protein interactions by affinity-directed mass spectrometry.

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.

Amino Acid Sequence↗
PubMed1996

Probing the chemical basis of binding activity in an SH3 domain by protein signature analysis.

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.

Amino Acid Sequence↗
PubMed1995

Detection of synthetic protein isomers and conformers by electrospray mass spectrometry.

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.

Amino Acid Sequence↗
PubMed1994

Synthesis of proteins by native chemical ligation.

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.

Amino Acid Sequence↗