Search PubMed⌕ Search

Biomedical subjects

R N Perham

Publications and source records attributed to R N Perham.

At least 19 recordsLinked to original sources
PubMed1995

Altering kinetic mechanism and enzyme stability by mutagenesis of the dimer interface of glutathione reductase.

In wild-type glutathione reductase from Escherichia coli residues Val421 and Ala422 are located in an alpha-helix in a densely packed and hydrophobic region of the dimer interface, with their side chains packed against those of residues Ala422' and Val421' in the second subunit. A series of mutant glutathione reductases was constructed in which the identities of the residues at positions 421 and 422 were changed. Mutations were designed so as to present like charges (mutants Val421-->Glu:Ala422-->Glu and Val421-->Lys:Ala422-->Lys) or opposite charges (mutant Val421-->Lys:Ala422-->Glu) across the dimer interface to assess the role of electrostatic interactions in dimer stability. A fourth mutant (Val421-->His:Ala422-->His) was also constructed to investigate the effects of introducing a potentially protonatable bulky side chain into a crowded region of the dimer interface. In all cases, an active dimeric enzyme was found to be assembled but each mutant protein was thermally destabilized. A detailed steady-state kinetic analysis indicated that each mutant enzyme no longer displayed the Ping Pong kinetic behaviour associated with the wild-type enzyme but exhibited what was best described as a random bireactant ternary complex mechanism. This leads, depending on the chosen substrate concentration, to apparent sigmoidal, hyperbolic or complex kinetic behaviour. These experiments, together with others reported previously, indicate that simple mutagenic changes in regions distant from the active site can lead to dramatic switches in steady-state kinetic mechanism.

Alanine↗
PubMed1995

Three-dimensional structure of a lipoyl domain from the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The structure of a lipoyl domain from the pyruvate dehydrogenase multienzyme complex of Escherichia coli has been determined by means of nuclear magnetic resonance spectroscopy. A total of 549 nuclear Overhauser effect distance restraints, 52 phi torsion angle restraints and 16 slowly exchanging amide protons were employed as input for the structure calculations. These were performed using a combined distance geometry-simulated annealing strategy. The domain is a hybrid between the N and C-terminal halves of the first and third lipoyl domains, respectively, of the dihydrolipoyl acetyltransferase component of the E. coli multienzyme complex, representing residues 1 to 33 and 238 to 289 (wild-type numbering). The lipoyl-lysine residue was also replaced by glutamine. Nonetheless, its structure, two four-stranded beta-sheets forming a flattened beta-barrel, closely resembles that of the lipoyl domain from the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus determined previously. As before, the lipoylation site is physically exposed in a tight turn in one of the beta-sheets, and the N and C-terminal residues are close together at the other end of the molecule in adjacent strands of the other beta-sheet. Another prominently conserved feature of the structure is the 2-fold axis of quasi-symmetry relating the N and C-terminal halves of the domain. Consistent with the high level of sequence similarity between lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes from many different sources, these results confirm that all lipoyl domains are likely to have closely related structures.

Acetyltransferases↗
PubMed1995

Interaction of component enzymes with the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: stoichiometry and specificity in self-assembly.

The interaction between the pyruvate decarboxylase (E1) component and a di-domain (lipoyl domain plus peripheral subunit-binding domain) from the dihydrolipoyl acetyltransferase (E2) component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex was investigated. Only 1 mol of di-domain (binding domain) was bound to 1 mol of heterotetrameric E1 (alpha 2 beta 2) and the binding was without effect on the kinetic activity of E1. Similarly, the di-domain bound to separate E1 beta subunits at a maximal polypeptide chain ratio of 1:2, but no detectable interaction was found with the E1 alpha subunit. However, addition of the monomeric E1 alpha subunit to an E1 beta-di-domain complex generated a fully functional E1 (alpha 2 beta 2)-di-domain complex, indicating that the E1 beta subunit plays the critical part in binding the E1 component to the di-domain and suggesting that no chaperonin is needed in vitro to promote the assembly of the three separate proteins. Mixing the E1 and dihydrolipoyl dehydrogenase (E3) components in the presence of di-domain revealed that E1 and E3 cannot bind simultaneously to the same molecule of di-domain, a new feature of the assembly pathway and an important factor in determining the ultimate structure of the assembled enzyme complex.

Binding Sites↗
PubMed1995

Matching electrostatic charge between DNA and coat protein in filamentous bacteriophage. Fibre diffraction of charge-deletion mutants.

The virion of Ff (fd, f1, M13) filamentous bacteriophage consists of a long tube of coat protein subunits in a shingled, helical array, surrounding a genome of circular single-stranded DNA. Modified fd virions have been generated by a mutation (K48A) that removes one positive charge from each coat protein subunit in the C-terminal region of the polypeptide chain facing the DNA. The number of nucleotides in the mutant DNA is unchanged, but the K48A virions are 35% longer than wild-type. We have measured the X-ray diffraction attributable to single virions in hydrated gels of wild-type and K48A bacteriophages. Most of the diffraction pattern shows no significant difference between wild-type and K48A. Since the DNA is only about 12% by weight of the wild-type virion, the diffraction pattern is dominated by the protein contribution, and the absence of significant differences indicates that there are no significant changes in the symmetry or structure of the protein coat. But there is a change in the diffraction pattern in a region where the DNA and protein contributions are comparable. The diffraction pattern of the K48A mutant shows an increase in intensity of one of the weaker equatorial peaks, relative to wild-type, in a region where the protein contribution has negative sign but the DNA contribution has positive sign. This is consistent with a decrease in the ratio of DNA:protein per unit length of the K48A mutant. The results support the view that the protein forms a sheath lined with positive charges interacting electrostatically and non-specifically with a negatively charged DNA core of matching charge density. The lower positive charge density lining the capsid in the K48A mutant means that correspondingly fewer nucleotides can be packaged per coat protein subunit, which in turn requires an elongation of the DNA inside the virion. A longer virion is thus required to package the same amount of DNA. Within the error of measurement, the number of positive charges on the protein interacting with the DNA is the same in K48A as in the wild-type, despite the fact that the mutant is 35% longer than the wild-type.

Capsid↗
PubMed1995

Engineering a peptide epitope display system on filamentous bacteriophage.

The genome of bacteriophage fd has been engineered to allow foreign amino acid sequences to be displayed in the exposed N-terminal segment of the major coat protein in the virus particle: small peptides can be encoded directly; larger peptides are encoded in hybrid virions, in which wild-type coat protein subunits are interspersed with coat proteins displaying the foreign peptides. Biophysical techniques, such as X-ray diffraction, indicate that the inclusion of the peptides can be achieved without significant disturbance to the helical parameters that define the protein-protein interactions in the assembled virion and the exposure of the peptides can be verified by analysing the susceptibility to attack by proteolytic enzymes. Peptide sequences from the V3 loop of the surface glycoprotein gp120 of HIV-1 strain MN (HIV-1MN) displayed in this way are remarkably effective structural mimics of the natural epitope. They are recognised by human HIV antisera and evoke high titres of virus-neutralizing antibodies in mice. Antibody production is stimulated by simultaneous inoculation with T cell epitopes similarly displayed on filamentous bacteriophage. The bacteriophage display system offers a powerful means of studying the immunological recognition of proteins. The specificity of the immune response, the ability to recruit helper T cells, the lack of need for external adjuvants and the structural mimicry of defined peptide epitopes, suggest that it will also be an inexpensive and simple route to the production of effective vaccines.

Amino Acid Sequence↗
PubMed1994

Reductive and oxidative half-reactions of glutathione reductase from Escherichia coli.

Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH and has a redox active disulfide and an FAD cofactor in each monomer. In the reductive half-reaction, FAD is reduced by NADPH and electrons pass from the reduced flavin to the redox active disulfide. The oxidative half-reaction is dithiol-disulfide interchange between the enzyme dithiol and glutathione disulfide. We have investigated the reductive and oxidative half-reactions using wild-type glutathione reductase from Escherichia coli and in an altered form of the enzyme in which the active site acid-base catalyst, His439, has been changed to an alanine residue (H439A). H439A has 0.3% activity in the NADPH/GSSG assay. The replacement affects both the oxidative half-reaction, as expected, and the reductive half-reaction--specifically, the passage of electrons from reduced flavin to the disulfide. Reduction of H439A by NADPH allows direct observation of flavin reduction. The NADPH-FAD charge transfer complex is formed in the dead time. Reduction of FAD, at a limiting rate of 250 s-1, is observed as a decrease at 460 nm and an increase at 670 nm (FADH(-)-NADP+ charge transfer). Subsequent passage of electrons from FADH- to the disulfide (increase at 460 nm and a decrease at 670 nm) is very slow (6-7 s-1) and concentration independent in H439A. The monophasic oxidative half-reaction is very slow, as expected for reduced H439A.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1994

Structural mimicry and enhanced immunogenicity of peptide epitopes displayed on filamentous bacteriophage. The V3 loop of HIV-1 gp120.

The principal neutralizing determinant of the human immunodeficiency virus type 1 (HIV-1) is an intra-chain disulphide-bridged loop, designated V3, in the third hypervariable region of the surface glycoprotein gp120. Peptide sequences from the V3 loop of gp120 from HIV-1 strain MN (HIV-1MN) were engineered into the N-terminal region of the major coat protein of filamentous bacteriophage fd, leading to their display in multiple copies on the surface of the bacteriophage virion. Peptides displayed in this way were shown to be remarkably effective structural mimics of the natural epitope. They were recognised by human HIV antisera and evoked high titres of antibodies in mice, which cross-reacted with other strains of HIV and were capable of neutralizing the virus. In addition, antibody production could be stimulated by simultaneous inoculation with T-cell epitopes similarly displayed on filamentous bacteriophage. The bacteriophage display system offers a powerful means of studying the immunological recognition of proteins and is a promising vaccine model.

Amino Acid Sequence↗
PubMed1994

Structure of the NADPH-binding motif of glutathione reductase: efficiency determined by evolution.

The role of the second glycine residue (Gly-176) of the conserved GXGXXA "fingerprint" motif in the NADPH-binding domain of Escherichia coli glutathione reductase has been studied by means of site-directed mutagenesis. This glycine residue occurs at the N-terminus of the alpha-helix in the beta alpha beta fold that characterizes the dinucleotide-binding domain, in close proximity to the pyrophosphate bridge of the bound coenzyme. Introducing an alanine residue (G176A), the minimum possible change, at this position virtually inactivated the enzyme, as did the introduction of valine, leucine, isoleucine, glutamic acid, histidine, or arginine residues. Only the replacement by serine--a natural substitute for this glycine residue in some forms of mercuric reductase, a related flavoprotein disulfide oxidoreductase--produced a mutant enzyme (G176S) that retained significant catalytic activity. It is conceivable that this is due to a favorable hydrogen bond being formed between the serine hydroxyl and a pyrophosphate oxygen atom. In most of the mutant enzymes, the Km for NADPH was substantially greater than that found for wild-type glutathione reductase, as expected, but this was accompanied by an unexpected decrease in the Km for GSSG. The latter can be explained by the observation that the reduction of the enzyme by NADPH, the first half-reaction of the ping-pong mechanism, had become a rate-limiting step of the overall reaction catalyzed, albeit poorly, by the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Expression in Escherichia coli of genes encoding the E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex of Bacillus stearothermophilus and assembly of a functional E1 component (alpha 2 beta 2) in vitro.

The E1 alpha and E1 beta subunits of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus were produced from two genes overexpressed separately in Escherichia coli. A functional E1 enzyme was generated from disrupted mixtures of cells containing the separately overexpressed E1 alpha and E1 beta genes. The purified E1 enzyme exhibited an apparent molecular mass of 150,000 Da, consistent with an alpha 2 beta 2 structure. The Km for pyruvate and kcat (30 degrees C) were found to be 0.9 +/- 0.2 microM and 0.47 +/- 0.03 s-1, respectively. The purified E1 alpha subunit existed as a monomer (42,000 Da), whereas the E1 beta subunit existed mainly (95%) in a tetrameric form (145,000 Da). Mixing equimolar amounts of the pure recombinant E1 alpha and E1 beta subunits in vitro generated a functional E1 enzyme with a molecular mass and an E1 activity similar to those of the E1(alpha 2 beta 2) enzyme purified from disrupted mixtures of cells containing individually expressed subunits. Mixing individual subunits in vitro with one of the subunits in excess resulted in complete assembly of the lesser subunit into the intact E1 (alpha 2 beta 2) enzyme. Thus, no chaperonin is needed in vitro to promote the assembly of the separate subunits to form the E1 component of the pyruvate dehydrogenase multienzyme complex of B. stearothermophilus.

Base Sequence↗
PubMed1994

Solubilizing buried domains of proteins: a self-assembling interface domain from glutathione reductase.

In the dimeric glutathione reductase (GR) from Escherichia coli, the interface domain is largely surrounded by the other three domains in each subunit of the protein. Subgenes encoding three forms of the interface domain have been expressed in E. coli and the products purified from inclusion bodies: INT is the excised interface domain, as it is found in native GR; INTN and INTFN are variants carrying exchanges of surface residues in what would have been hydrophobic contact regions with other neighboring domains. The isolated INT domain was found to be a soluble and folded protein, but it was isolated as a mixture of the dimer and at least two species of higher molecular weight. The latter were believed to arise by further association of the dimer via the newly exposed and unsatisfied hydrophobic contact regions. In the variant INTN, three hydrophobic residues normally involved in the contact with the NADPH-binding domain in GR were replaced. This partly suppressed the further aggregation of the dimers. However, continued aggregation at high protein concentrations suggested that at least one further site of unwanted aggregation was still present. After four additional amino acid replacements in the region normally in contact with the FAD-binding domain, the resulting variant INTFN exhibited no unspecific aggregation, even at concentrations as high as 3.2 mg/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Structural dependence of post-translational modification and reductive acetylation of the lipoyl domain of the pyruvate dehydrogenase multienzyme complex.

The lipoyl domain of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complex is recognized specifically by the lipoylating enzyme(s) in the cell and by the pyruvate dehydrogenase (E1) component in the parent complex. Highly conserved aspartic acid and alanine residues flank the lipoyl-lysine residue, on the N and C-terminal sides, respectively, in the sharp beta-turn in which the lipoyl-lysine residue is prominently displayed. A sub-gene encoding the lipoyl domain of the Bacillus stearothermophilus pyruvate dehydrogenase complex was subjected to mutagenesis in the vector M13mp18. Aspartic acid 41 was changed to glutamic acid (D41E), alanine (D41A) and lysine (D41K), and alanine 43 was changed to methionine (A43M), lysine (A43K) and glutamic acid (A43E). The double mutations D41KK42A and D41MA43M were also made. All mutant domains were capable of being lipoylated, apart from the D41KK42A domain where the lipoyl-lysine had been moved round the beta-turn by one position towards the N terminus. Neither the D41K nor the A43K mutants showed any doubly lipoylated domain and the single lipoyl group was found attached only to the correct lysine residue. Accurate positioning of the lipoyl-lysine in the beta-turn is thus an essential cue for lipoylation, but the conserved aspartic acid and alanine residues are not necessary for the domain to be recognized by the lipoylating enzyme(s). No biotinylation of the D41MA43M mutant domain was observed, although the sequence motif MKM is highly conserved as the biotinylation site in the structurally homologous biotinyl domain of biotin-containing enzymes. The mutations at the aspartic acid 41 position all lowered the rate of reductive acetylation of the lipoyl domain by the E1 component of the pyruvate dehydrogenase complex, as did the mutations A43E and A43K. The A43M mutant was reductively acetylated at the same rate as the wild-type domain. Thus, both the alanine and aspartic acid residues are important for recognition of the domain by E1, but there is no absolute dependence on retention of the sequence surrounding the lipoyl-lysine residue.

Acetylation↗
PubMed1994

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus: preparation and characterization of its binding to the dihydrolipoyl dehydrogenase component.

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli. The domain was characterized by N-terminal sequence analysis, electrospray m.s. and c.d. spectroscopy. It was found to be identical in all respects to a chemically synthesized peptide of the same sequence. The association of the di-domain and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated. As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.

Amino Acid Sequence↗
PubMed1994

Anatomy of an engineered NAD-binding site.

The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of 16.8%), of the wild-type enzyme ligated with NADP (2.0 A, 20.8%), of the NAD-dependent mutant (1.74 A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD-binding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate.

Binding Sites↗
PubMed1994

Structural aspects of biomolecular recognition and self-assembly.

Proteins are capable of fulfilling two important features of any likely system of bioelectronics: the ability to recognise other molecules with exquisite specificity, and the ability to self-assemble, in vivo and in vitro, to generate an astonishing variety of three-dimensional structures. Much current work is aimed at the redesign of existing proteins, either as an end in itself or as a means of developing the knowledge-base necessary for the ab initio design of novel proteins. This type of study has been greatly facilitated by the discovery of the modular or domain structure of many proteins, leading to concepts of protein manipulation as a kind of molecular Lego.

Protein Engineering↗
PubMed1993

Immunological properties of foreign peptides in multiple display on a filamentous bacteriophage.

The genome of bacteriophage fd has been engineered to permit construction of hybrid virus particles in which the wild-type major coat protein (gpVIII) subunits were interspersed with coat proteins displaying one or other of two foreign peptides (fdMAL1, sequence NANPNANPNANP or fdMAL2, sequence NDDSYIPSAEKI) in the exposed N-terminal segments [Greenwood et al., J. Mol. Biol. 220 (1991) 821-827]. These sequences represent major antigenic determinants of the circumsporozoite protein of the malaria parasite, Plasmodium falciparum. The peptide epitopes in the hybrid bacteriophages were found to be strongly immunogenic in four different strains of mice without the use of external adjuvants, and the antibodies (Ab) were highly specific to the individual epitopes in ELISA assays. When tested in nude (nu/nu) and heterozygote (nu +/-) BALB/c mice, the immune response was found to be T-cell dependent and to undergo class-switching from IgM to IgG. Proliferation assays of T-cells taken from lymph nodes of BALB/c mice injected with bacteriophage particles in the presence or absence of Freund's complete adjuvant indicated no difference in the immune response. This way of generating Ab against peptide epitopes is simpler and much less expensive than the conventional method of peptide synthesis and coupling to a carrier protein for injection. The specificity of the immune response, the ability to recruit helper T-cells and the lack of need for external adjuvants suggest that it will also be an inexpensive and simple route to the production of effective vaccines.

Amino Acid Sequence↗
PubMed1993

Structural differences between wild-type NADP-dependent glutathione reductase from Escherichia coli and a redesigned NAD-dependent mutant.

NAD and NADP are ubiquitous coenzymes in biological redox reactions. They have distinct metabolic functions, yet they differ only by an additional phosphate group esterified at the 2'-hydroxyl group of the AMP moiety of NADP. The natural specificity of Escherichia coli glutathione reductase for NADP has previously been converted into a marked preference for NAD by introducing seven point mutations into the beta alpha beta-fold of the NADP-binding domain of the protein based on the known structure of the human enzyme. Among them was the replacement of Ala179 by glycine (A179G) in the alpha-helix of the fold, a change suggested by a difference in a sequence fingerprint previously found in the dinucleotide-binding domains of a number of dehydrogenases. Although this position is at a distance of 10 A from the bound 2'-phosphate group of NADP in glutathione reductase, the A179G mutation was found to be synergistic and beneficial. We have now carried out X-ray crystallographic analyses of the NAD-dependent mutant without and with bound NADH. A comparison of the structures of the mutant and wild-type enzymes reveals a flip of the peptide bond between Gly174 and Ala175 such that the side-chain of another introduced amino acid, Glu197, is fixed and can participate in binding the adenine ribose of NAD, thereby contributing to the ability of the mutated enzyme to exert its selectivity for the "wrong" coenzyme.

Amino Acid Sequence↗