Semisynthesis of a functional K+ channel.
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Amino Acid Sequence↗
Biomedical subjects
Tom W Muir
Publications and source records attributed to Tom W Muir.
Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein-intein junction.
Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. Although we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how these are catalyzed and regulated is less well developed. In the current study, a combination of NMR spectroscopy and segmental isotopic labeling has been used to study the structure of an active protein splicing precursor, corresponding to an N-extein fusion of the Mxe GyrA intein. The (1)J(NC') coupling constant for the (-1) scissile peptide bond at the N-extein-intein junction was found to be approximately 12 Hz, which indicates that this amide is highly polarized, perhaps because of nonplanarity. Additional mutagenesis and NMR studies indicate that conserved box B histidine residue is essential for catalysis of the first step of splicing and for maintaining the (-1) scissile bond in its unusual conformation. Overall, these studies support the "ground-state destabilization" model as part of the mechanism of catalysis.
Isotopes↗
Segmental isotopic labeling for structural biological applications of NMR.
This chapter describes the preparation of precursor domains for the formation of multidomain segmentally labeled proteins by protein ligation.
Cloning, Molecular↗
"The splice is right": how protein splicing is opening new doors in protein science.
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Biotechnology↗
Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo.
Protein splicing is a naturally occurring process in which an intervening intein domain excises itself out of a precursor polypeptide in an autocatalytic fashion with concomitant linkage of the two flanking extein sequences by a native peptide bond. We have recently reported an engineered split VMA intein whose splicing activity in trans between two polypeptides can be triggered by the small molecule rapamycin. In this report, we show that this conditional protein splicing (CPS) system can be used in mammalian cells. Two model constructs harboring maltose-binding protein (MBP) and a His-tag as exteins were expressed from a constitutive promoter after transient transfection. The splicing product MBP-His was detected by Western blotting and immunoprecipitation in cells treated with rapamycin or a nontoxic analogue thereof. No background splicing in the absence of the small-molecule inducer was observed over a 24-h time course. Product formation could be detected within 10 min of addition of rapamycin, indicating the advantage of the posttranslational nature of CPS for quick responses. The level of protein splicing was dose dependent and could be competitively attenuated with the small molecule ascomycin. In related studies, the geometric flexibility of the CPS components was investigated with a series of purified proteins. The FKBP and FRB domains, which are dimerized by rapamycin and thereby induce the reconstitution of the split intein, were fused to the extein sequences of the split intein halves. CPS was still triggered by rapamycin when FKBP and FRB occupied one or both of the extein positions. This finding suggests yet further applications of CPS in the area of proteomics. In summary, CPS holds great promise to become a powerful new tool to control protein structure and function in vitro and in living cells.
Animals↗
Protein semi-synthesis in living cells.
Incorporation of chemical probes into proteins is a powerful way to elucidate biological processes and to engineer novel function. Here we describe an approach that allows ligation of synthetic molecules to target proteins in an intracellular environment. A cellular protein is genetically tagged with one-half of a split intein. The complementary half is linked in vitro to the synthetic probe, and this fusion is delivered into cells using a transduction peptide. Association of the intein halves in the cytosol triggers protein trans-splicing, resulting in the ligation of the probe to the target protein through a peptide bond. This process is specific and applicable to cytosolic and integral membrane proteins. The technology should allow cellular proteins to be elaborated with a variety of abiotic probes.
Animals↗
A modular cross-linking approach for exploring protein interactions.
A method is described for the elucidation of protein-protein interactions using novel cross-linking reagents and mass spectrometry. The method incorporates (1) a modular solid-phase synthetic strategy for generating the cross-linking reagents, (2) enrichment and digestion of cross-linked proteins using microconcentrators, (3) mass spectrometric analysis of cross-linked peptides, and (4) comprehensive computational analysis of the cross-linking data. This integrated approach has been applied to the study of cross-linking between the components of the heterodimeric protein complex negative cofactor 2.
Acrylic Resins↗
Semisynthesis of proteins by expressed protein ligation.
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.
Amino Acids↗
Toward fully synthetic N-linked glycoproteins.
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Acylation↗
Turning virulence on and off in staphylococci.
The progress made in a multidisciplinary research programme designed to elucidate the molecular basis of the interaction of Staphylococcus aureus secreted autoinducing peptides (AIPs) with their respective cell surface receptors is reviewed.
Bacterial Proteins↗
Chemical signaling among bacteria and its inhibition.
Generations of chemists and biologists have conducted research on natural products and other metabolites produced by bacteria and other microorganisms. This has led to an explosion in knowledge concerning the mechanism by which such natural products are made, ultimately allowing custom redesign of many of these molecules for increased potency and selectivity as therapeutic drugs. Along the way, scientists have begun to appreciate that the bacterial world is teeming with life on a scale hardly conceivable, with constant communication within the bacterial world and with outside neighbors, such as plants and mammals. Only in recent years have some of the signaling molecules that comprise these elaborate forms of communication been characterized in any sort of chemical detail, which has in turn peaked interest in the intricate biology of this micro-world and its interactions with the macro-world.
Bacteria↗
Semisynthesis and folding of the potassium channel KcsA.
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.
Amino Acid Sequence↗
Protein splicing triggered by a small molecule.
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.
Blotting, Western↗
Key determinants of receptor activation in the agr autoinducing peptides of Staphylococcus aureus.
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.
Amino Acid Sequence↗
Autoregulation of a bacterial sigma factor explored by using segmental isotopic labeling and NMR.
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.
Amino Acid Sequence↗
Efficient semisynthesis of a tetraphosphorylated analogue of the Type I TGFbeta receptor.
[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.
Activin Receptors, Type I↗
Recent advances in the application of expressed protein ligation to protein engineering.
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.
Amino Acids↗
Reversible and specific extracellular antagonism of receptor-histidine kinase signaling.
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.
Amino Acid Sequence↗