Search PubMed⌕ Search

Biomedical subjects

R N Perham

Publications and source records attributed to R N Perham.

At least 37 records · Page 2Linked to original sources
PubMed1999

Interaction of the globular domains of pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli.

Gene 3 protein (pIII), a minor coat protein at one end of the filamentous bacteriophage fd, is involved in initiating the infection by the virus of Escherichia coli cells that display an F-pilus. Infection is thought to start with the adsorption of the D2 domain of pIII to the tip of the pilus, retraction of the pilus, and penetration of the E. coli cell membrane mediated by an interaction between the D1 domain of pIII and the Tol protein complex in the membrane. A subgene encoding the pIII-D1D2 di-domain was created, and the subgene was successfully overexpressed in E. coli cells. Domains D1 and D2 were separated after limited proteolysis of a modified pIII-D1D2 (designated pIII-D1D2.trp) into which two tryptic cleavage sites were introduced at appropriate points. The purified pIII-D1D2 di-domain and pIII-D2 domain were able to bind to the F-pilus, competing with the wild-type pIII and delaying infection by the intact filamentous phage. The pIII-D1 domain was unable to bind to the F-pilus by this criterion. This provides conclusive evidence that the pIII-D2 domain is responsible for the adsorption to the tip of the F-pilus and can achieve this in the absence of domain D1, opening the way to identifying the molecular basis of the interaction of pIII-D2 with the pilus.

Binding Sites↗
PubMed1999

Self-assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus.

The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was reconstituted in vitro from recombinant proteins derived from genes over-expressed in Escherichia coli. Titrations of the icosahedral (60-mer) dihydrolipoyl acetyltransferase (E2) core component with the pyruvate decarboxylase (E1, alpha2beta2) and dihydrolipoyl dehydrogenase (E3, alpha2) peripheral components indicated a variable composition defined predominantly by tight and mutually exclusive binding of E1 and E3 with the peripheral subunit-binding domain of each E2 chain. However, both analysis of the polypeptide chain ratios in complexes generated from various mixtures of E1 and E3, and displacement of E1 or E3 from E1-E2 or E3-E2 subcomplexes by E3 or E1, respectively, showed that the multienzyme complex does not behave as a simple competitive binding system. This implies the existence of secondary interactions between the E1 and E3 subunits and E2 that only become apparent on assembly. Exact geometrical distribution of E1 and E3 is unlikely and the results are best explained by preferential arrangements of E1 and E3 on the surface of the E2 core, superimposed on their mutually exclusive binding to the peripheral subunit-binding domain of the E2 chain. Correlation of the subunit composition with the overall catalytic activity of the enzyme complex confirmed the lack of any requirement for precise stoichiometry or strict geometric arrangement of the three catalytic sites and emphasized the crucial importance of the flexibility associated with the lipoyl domains and intramolecular acetyl group transfer in the mechanism of active-site coupling.

Binding, Competitive↗
PubMed1998

Analysis of X-ray diffraction from fibres of Pf1 Inovirus (filamentous bacteriophage) shows that the DNA in the virion is not highly ordered.

X-ray fibre diffraction patterns of well-aligned Pf1 filamentous bacteriophage show sharp layer-lines attributable to an ordered helical array of protein subunits. Electron density maps calculated from the intensity on these layer-lines show no evidence for DNA following the symmetry of the protein, nor is there evidence on the diffraction patterns for the additional layer-lines expected if ordered DNA follows a symmetry different from that of the protein. We conclude that the interactions between DNA and protein in the Pf1 virion, like those in the Ff virion, are delocalized rather than specific, and the DNA structure in the virion is less regular than the protein structure. This conclusion has implications for the process of virion assembly, and we suggest a possible model for the change in the viral DNA symmetry as the DNA is passed to the virion from the intracellular complex with the viral gene 5 single-stranded DNA-binding protein.

Capsid↗
PubMed1998

Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro.

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta. This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.

Genes, Bacterial↗
PubMed1998

Factors limiting display of foreign peptides on the major coat protein of filamentous bacteriophage capsids and a potential role for leader peptidase.

Many small peptides can be displayed on every copy of the major coat protein in recombinant filamentous bacteriophages but larger peptides can only be accommodated in hybrid virions mixed with wild-type protein subunits. A peptide insert of 12 residues capable of display at high copy number in a hybrid virion was found to be incapable of supporting recombinant virion assembly, a defect that could not be overcome by over-expressing leader peptidase in the same Escherichia coli cell. In contrast, over-expressing leader peptidase did increase the copy number of two 9-residue peptides that were poorly incorporated into hybrid virions. The factors that limit peptide display are varied and not restricted to the early stages of viral assembly.

Amino Acid Sequence↗
PubMed1998

Selectivity of post-translational modification in biotinylated proteins: the carboxy carrier protein of the acetyl-CoA carboxylase of Escherichia coli.

Biotin-dependent enzymes contain a biotinyl-lysine residue in a conserved sequence motif, MKM, located in a surface hairpin turn in one of the two beta-sheets that make up the domain. A sub-gene encoding the 82-residue C-terminal biotinyl domain from the biotin carboxy carrier protein of acetyl-CoA carboxylase from Escherichia coli as a fusion protein with glutathione S-transferase was created and over-expressed in E. coli. The biotinyl domain was readily released by cleavage with thrombin. Five mutant domains were created in which the conserved MKM motif was systematically replaced: by MAK and KAM, in which the target lysine is moved one place; by KKM and MKK, in which a second potential site for biotinylation is introduced; and by DKA, the motif found in the correspondingly conserved site of lipoylation in the structurally related lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes. No biotinylation of the MAK or KAM mutants was observed in vivo or by purified biotinyl protein ligase in vitro; in the KKM and MKK mutants, only one lysine residue, presumed to be that in its native position in the hairpin turn, was found to be biotinylated in vivo and in vitro. The DKA mutant was not biotinylated in vivo, but was partly lipoylated and octanoylated. It was also a poor substrate for lipoylation in vitro catalysed by the E. coli lipoyl protein ligase encoded by the lplA gene. The flanking sequence in the MKM motif is important, but not crucial, and appears to have been conserved in part to be compatible with the subsequent carboxylation reactions of biotin-dependent enzymes. The DKA motif, displayed in the hairpin loop, is sufficient to address lipoylation in E. coli but probably by a pathway different from that mediated by the lplA-dependent ligase. The recognition of the structurally homologous lipoyl and biotinyl domains by the appropriate ligase evidently has a major structural component to it, notably the positioning of the target lysine residue in the exposed hairpin loop, but there appear to be additional recognition sites elsewhere on the domains.

Acetyl-CoA Carboxylase↗
PubMed1998

Structure of the capsid of Pf3 filamentous phage determined from X-ray fibre diffraction data at 3.1 A resolution.

We have recorded X-ray diffraction patterns at 3.1 A resolution from magnetically aligned fibres of the Pf3 strain of filamentous bacteriophage (Inovirus). The patterns are similar to patterns from the higher-temperature form of the Pf1 strain, indicating that the Pf3 and Pf1 virions have the same helix symmetry and similar protein subunit shape. This is of particular interest, given that the primary structures of the two protein subunits are quite different; and the nucleotide/protein subunit ratio in the Pf3 virion is more than twice that in Pf1, indicating important differences in DNA packaging. We have built a molecular model of the Pf3 protein capsid based on the model of Pf1, and refined it against the diffraction data using simulated annealing. The refinement confirms that the two structures are similar, which may reflect a fundamental motif of alpha-helix packing. However, there are some differences between the structures: the Pf3 subunit appears to be completely alpha-helical, beginning at the N terminus, whereas the first few residues of the Pf1 subunit are not helical; and the structure of the C-terminal region of the Pf3 subunit at the inner surface of the tubular capsid indicates that DNA/protein interactions in this virion may involve both aromatic side-chains and positively charged side-chains, whereas those in the Pf1 virion involve predominantly only the latter. In the course of this work, we have developed new approaches to refinement and validation of helical structures with respect to continuous transform fibre diffraction data.

Amino Acid Sequence↗
PubMed1998

Flavin fluorescence dynamics and photoinduced electron transfer in Escherichia coli glutathione reductase.

Time-resolved polarized flavin fluorescence was used to study the active site dynamics of Escherichia coli glutathione reductase (GR). Special consideration was given to the role of Tyr177, which blocks the access to the NADPH binding-site in the crystal structure of the enzyme. By comparing wild-type GR with the mutant enzymes Y177F and Y177G, a fluorescence lifetime of 7 ps that accounts for approximately 90% of the fluorescence decay could be attributed to quenching by Y177. Based on the temperature invariance for this lifetime, and the very high quenching rate, electron transfer from Y177 to the light-excited isoalloxazine part of flavin adenine dinucleotide (FAD) is proposed as the mechanism of flavin fluorescence quenching. Contrary to the mutant enzymes, wild-type GR shows a rapid fluorescence depolarization. This depolarization process is likely to originate from a transient charge transfer interaction between Y177 and the light-excited FAD, and not from internal mobility of the flavin, as has previously been proposed. Based on the fluorescence lifetime distributions, the mutants Y177F and Y177G have a more flexible protein structure than wild-type GR: in the range of 223 K to 277 K in 80% glycerol, both tyrosine mutants mimic the closely related enzyme dihydrolipoyl dehydrogenase. The fluorescence intensity decays of the GR enzymes can only be explained by the existence of multiple quenching sites in the protein. Although structural fluctuations are likely to contribute to the nonexponential decay and the probability of quenching by a specific site, the concept of conformational substates need not be invoked to explain the heterogeneous fluorescence dynamics.

Binding Sites↗
PubMed1998

Three-dimensional structure of the major autoantigen in primary biliary cirrhosis.

BACKGROUND & AIMS: Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies in patients' serum. The major autoantigen, recognized by antibodies from > 95% of patients with PBC, has been identified as the E2 component (E2p) of the pyruvate dehydrogenase multienzyme complex. Immunodominant sites on E2p have been localized to the inner of the two lipoyl domains, where the essential cofactor lipoic acid is attached covalently. The aim of this study was to determine the three-dimensional structure of the inner lipoyl domain of human E2p. METHODS: The domain was expressed in Escherichia coli; after purification, its structure was analyzed using nuclear magnetic resonance spectroscopy. RESULTS: The structure of the lipoyl domain from human E2p was determined, and the implications of the structure for autoimmune recognition were assessed. CONCLUSIONS: Knowledge of the structure further defines the major epitope and may help in the design of antigen-specific immunotherapy for treatment of PBC.

Amino Acid Sequence↗
PubMed1997

The catalytic domain of dihydrolipoyl acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Expression, purification and reversible denaturation.

A sub-gene encoding the catalytic (acetyltransferase) domain (E2pCD) comprising residues 173-427 of the dihydrolipoyl acetyltransferase (E2p) chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was expressed in Escherichia coli. The product assembled to form the characteristic icosahedral (60-mer) core structure with full catalytic activity. The Km values for dihydrolipoamide and acetyl-CoA were 1.2 mM and 13 microM, respectively. Dissociation of the icosahedral E2pCD into monomers by exposure to guanidine hydrochloride and the subsequent reassociation by gradual removal of the denaturing agent demonstrated the ability of the polypeptide chain to fold and reassemble in the absence of chaperonins.

Acetyl Coenzyme A↗
PubMed1997

NMR structure of the principal neutralizing determinant of HIV-1 displayed in filamentous bacteriophage coat protein.

An NMR approach for structure determination of short peptides displayed on the surface of filamentous bacteriophage virions is demonstrated using the hexapeptide GPGRAF that constitutes the principal neutralizing determinant of HIV-1. This peptide was inserted near the N terminus of the major coat protein of bacteriophage fd. NMR studies of the recombinant protein solubilized in detergent micelles showed that the inserted peptide adopts a double bend S-shaped conformation that is similar to the antibody-bound structure determined by X-ray crystallography. This indicates that a peptide displayed on the bacteriophage coat protein has an enhanced propensity to adopt a conformation similar to that found in the native protein from which it is derived. This approach may be generally applicable to the structure determination of peptide epitopes and other small peptides.

Capsid↗
PubMed1997

Simultaneous display of different peptides on the surface of filamentous bacteriophage.

We have developed a new system for producing hybrid virions of filamentous bacteriophage fd simultaneously displaying two different peptides by infecting cells harbouring a plasmid containing a modified gene VIII with an engineered bacteriophage carrying a second and different copy of a modified gene VIII. The simultaneous display of different peptides has many potential applications in exploring the immune response and studying protein-protein interaction.

Amino Acid Sequence↗
PubMed1997

Accessibility of peptides displayed on filamentous bacteriophage virions: susceptibility to proteinases.

The genome of the filamentous bacteriophage fd has been engineered so that small peptides can be inserted into the exposed N-terminal segment of pVIII, the major protein of the virus capsid. Most small peptides can be displayed on all 2700 copies of pVIII (a recombinant virion), but larger peptides can be displayed only in virions in which modified and wild-type proteins are intermingled (hybrid virions). Peptides displayed in this way are highly immunogenic and capable of interacting with receptors and other ligands. The physical accessibility of the displayed peptides was tested by examining their susceptibility to digestion with proteinases. Potential cleavage sites in peptides displayed on recombinant or hybrid virions were in general found to be accessible to trypsin and chymotrypsin; and the density of incorporation of peptides in the virion had no effect on the susceptibility to cleavage. However, peptide bonds towards the C-terminal end of an insert, located approximately 47 residues or fewer from the C-terminus of the coat protein, were protected from digestion, presumably because of their proximity to the bulk viral surface. These results have important implications for the design and optimization of peptide display systems using filamentous bacteriophages.

Amino Acid Sequence↗
PubMed1996

Competitive interaction of component enzymes with the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: kinetic analysis using surface plasmon resonance detection.

The interactions of the peripheral enzymes (E1, a pyruvate decarboxylase, and E3, dihydrolipoyl dehydrogenase) with the core component (E2, dihydrolipoyl acetyltransferase) of the pyruvate dehydrogenase (PDH) multienzyme complex of Bacillus stearothermophilus have been analyzed using a biosensor based on surface plasmon resonance detection. A recombinant di-domain (lipoyl domain plus peripheral subunit-binding domain) from E2 was attached to the biosensor chip by means of the pendant lipoyl group. The dissociation constant (Kd) for the complex between the peripheral subunit-binding domain and E3 (5.8 x 10(-10) M) was found to be almost twice that for the complex with E1 (3.24 x 10(-10) M). This was due to differences in the rate constants for dissociation (kdiss); these were 1.06 x 10(-3) and 1.87 x 10(-3) s-1 for the complexes with E1 and E3, respectively, whereas the rate constants for association (kass) were identical (3.26 x 10(6) M-1 s-1). Separate studies using non-denaturing polyacrylamide gel electrophoresis confirmed the difference in affinity and demonstrated that E1 can rapidly displace E3 from an E3-di-domain complex and vice versa. The peripheral subunit-binding domain showed no detectable interaction with the E1 alpha subunit of E1 (alpha 2 beta 2) but exhibited a strong affinity for E1 beta (Kd = 8.5 x 10(-9) M), confirming that the E1 beta subunit is responsible for binding E1 to E2. These measurements introduce new features of potential importance into the assembly and mechanism of the multienzyme complex.

Binding Sites↗
PubMed1996

Three-dimensional structure of the lipoyl domain from the dihydrolipoyl succinyltransferase component of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli.

A sub-gene encoding the lipoyl domain of the dihydrolipoyl succinyltransferase polypeptide chain of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli was over-expressed and the protein was purified uniformly labelled with 15N. The three-dimensional structure of the domain was determined by means of nuclear magnetic resonance spectroscopy, based on 905 nuclear Overhauser effect inter-proton distance restraints, 42 phi torsion angle restraints and hydrogen bond restraints from 24 slowly exchanging amide protons. The structure of the 80-residue domain is that of a flattened beta-barrel surrounding a hydrophobic core in which Trp22 plays a central role in anchoring two four-stranded sheets together. The polypeptide backbone exhibits a 2-fold axis of quasi-symmetry, with the lipoylation site, Lys43, located at the tip of an exposed beta-turn in one beta-sheet and the N and C-terminal residues close together in space in the other beta-sheet. The atomic r.m.s. distribution about the mean coordinate is 0.46 A for the backbone atoms in the highly structured region and 0.88 A along the entire backbone (residues Ser1 to Asn80), including a less well-defined surface loop and the lipoyl-lysine beta-turn. The structure closely resembles that of the lipoyl domains from pyruvate dehydrogenase complexes, in accord with the existence of strongly conserved residues at critical positions in the domains. The structures of the lipoyl domains throw light on the requirements for the specificity of reductive acylation of their pendant lipoyl groups in the parent 2-oxo acid dehydrogenase complexes; an important aspect of the mechanisms underlying active site coupling and substrate channelling.

Acyltransferases↗
PubMed1996

Recognition of a surface loop of the lipoyl domain underlies substrate channelling in the pyruvate dehydrogenase multienzyme complex.

In the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus, the interaction between the pyruvate decarboxylase (E1p) component and the lipoyl domain of the dihydrolipoyl acetyltransferase (E2) component was investigated using a combination of site-directed mutagenesis and NMR spectroscopy. Residues 11 to 15 (EGIHE) of the lipoyl domain, part of a surface loop close in space to the beta-turn containing the lipoyl-lysine residue (position 42), were deleted or replaced. The mutant domains all retained their three-dimensional structures and ability to become lipoylated, but in the absence of the loop the lipoyl-lysine residue could no longer be reductively acetylated by E1p. A mutation (N40A) in the N- terminal part of the lipoyl-lysine hairpin showed that it is involved in recognition of the domain by E1p but other mutations in the loop (E15A) and close to the lipoyl-lysine hairpin (V44S, V45S and E46A) were without effect. The heteronuclear multiple quantum coherence NMR spectra of 15N-labelled lipoyl domain in the presence and absence of B. stearothermophilus E1p were recorded. Of the 85 amino acid residues in the lipoyl domain, 13 exhibited significant differences in chemical shift. These differences, most of which were associated with residues in the surface loop between positions 8 and 15 and in, or close to, the lipoyl-lysine hairpin, indicate that E1p makes contact with the lipoyl domain in these areas. The combined results of directed mutagenesis and NMR spectroscopy point to the surface loop as a major determinant of the interaction of lipoyl domain with E1p. The specificity of this essential interaction provides the molecular basis of substrate channelling in this, the first committed, step of the enzyme reaction mechanism.

Acetylation↗