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G C Roberts

Publications and source records attributed to G C Roberts.

At least 55 records · Page 3Linked to original sources
PubMed1997

A single mutation in cytochrome P450 BM3 changes substrate orientation in a catalytic intermediate and the regiospecificity of hydroxylation.

Phenylalanine 87 of Bacillus megaterium cytochrome P450 BM3, a residue close to the heme in the substrate binding pocket, has been replaced by alanine by site-directed mutagenesis. The substitution had no effect on the rate of hydroxylation of laurate and increased the affinity for laurate of both the intact enzyme and its heme domain by 2.6-6-fold in the ferric state. NMR paramagnetic relaxation measurements showed that in the initial ferric enzyme-substrate complex, where the substrate binds relatively far from the heme, the substitution had no effect on the position or orientation of the bound substrate. However, in the next intermediate in the catalytic cycle, the reduced enzyme, the position of the bound substrate was altered so that the terminal methyl group was 3.1 A from the iron in the mutant, compared to 5.1 A in the wild-type enzyme. Analysis of the products of the action of the enzyme on laurate and myristate showed that the mutant catalyzed hydroxylation almost exclusively at the omega position, in marked contrast to the wild-type enzyme, with which no hydroxylation at this position was observed.

Bacillus megaterium↗
PubMed1997

The influence of aspartate 26 on the tautomeric forms of folate bound to Lactobacillus casei dihydrofolate reductase.

The ternary complex of Lactobacillus casei dihydrofolate reductase (DHFR) with folate and NADP+ exists as a mixture of three interconverting forms (I, IIa and IIb) whose relative populations are pH dependent, with an effective pK of approx. 6. To investigate the role of Asp26 in this pH dependence we have measured the 13C chemical shifts of [2,4a,7,9-(13)C4]folate in its complex with the mutant DHFR Asp26 --> Asn and NADP+. Only a single form of the complex is detected and this has the characteristics of form I, an enol form with its N1 unprotonated. A study of the pH dependence of the 13C chemical shifts of DHFR selectively labelled with [4-(13)C]aspartic acid in its complex with folate and NADP+ indicates that no Asp residue has a pK value greater than 5.4. Two of the Asp CO2 signals appear as non-integral signals with chemical shifts typical of non-ionised COOH groups and with a pH dependence characteristic of the slow exchange equilibria previously characterised for signals in forms I and IIb (or IIa). It is proposed that the protonation/deprotonation controlling the equilibria involves the O4 position of the folate and that Asp26 influences this indirectly by binding in its CO2 form to the protonated N1 group of folate in forms I and IIa thus reducing the pK involving protonation at the O4 position to approx. 6. These findings indicate that, in forms I and IIa of the ternary complex, folate binds to DHFR in a very similar way to methotrexate.

Amino Acid Sequence↗
PubMed1997

Identification of the binding site on S100B protein for the actin capping protein CapZ.

The calcium-binding protein S100B binds to several potential target proteins, but there is no detailed information showing the location of the binding site for any target protein on S100B. We have made backbone assignments of the calcium-bound form of S100B and used chemical-shift changes in spectra of 15N-labeled protein to locate the site that binds a peptide corresponding to residues 265-276 from CapZ alpha, the actin capping protein. The largest chemical-shift changes are observed for resonances arising from residues around the C terminus of the C-terminal helix of S100B and residues Val-8 to Asp-12 of the N-terminal helix. These residues are close to but not identical to residues that have been identified by mutational analysis to be important in other S100 protein-protein interactions. They make up a patch across the S100B dimer interface and include some residues that are quite buried in the structure of calcium-free S100B. We believe we may have identified a binding site that could be common to many S100 protein-protein interactions.

Amino Acid Sequence↗
PubMed1997

1H, 15N and 13C NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450 reductase.

The FMN-binding domain of human NADPH-cytochrome P450 reductase, corresponding to exons 3-7, has been expressed at high level in an active form and labelled with 13C and 15N. Most of the backbone and aliphatic side-chain 1H, 15N and 13C resonances have been assigned using heteronuclear double- and triple-resonance methods, together with a semiautomatic assignment strategy. The secondary structure as estimated from the chemical shift index and NOE connectivities consists of six alpha-helices and five beta-strands. The global fold was deduced from the long-range NOEs unambiguously assigned in a 4D 13C-resolved HMQC-NOESY-HMQC spectrum. The fold is of the alternating alpha/beta type, with the five beta-strands arranged into a parallel beta-sheet. The secondary structure and global fold are very similar to those of the bacterial flavodoxins, but the FMN-binding domain has an extra short helix in place of a loop, and an extra helix at the N-terminus (leading to the membrane anchor domain in the intact P450 reductase). The experimental constraints were combined with homology modelling to obtain a structure of the FMN-binding domain satisfying the observed NOE constraints. Chemical shift comparisons showed that the effects of FMN binding and of FMN reduction are largely localised at the binding site.

Amino Acid Sequence↗
PubMed1997

Metabolism of MPTP by cytochrome P4502D6 and the demonstration of 2D6 mRNA in human foetal and adult brain by in situ hybridization.

1. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a thermal breakdown product of synthetic 'street' heroin, is known to cause Parkinson's Disease-like symptoms in man. 2. The mechanism of action of this neurotoxin is thought to involve activation by the monoamine oxidase B system and subsequent toxicity by inhibition of neuronal mitochondrial respiration. The manifestation of toxicity will be a balance between the rate of activation of this compound versus its rate of inactivation through metabolism by enzymes such as the cytochrome P450-dependent monooxygenases. 3. In this report we demonstrate that MPTP N-demethylation, a detoxification pathway, is catalysed by cytochrome P450 CYP2D6 and up to 40% of the hepatic metabolism is mediated by this enzyme. 4. Perhaps more importantly we also demonstrate by in situ hybridization that CYP2D6 is localized in the pigmented neurons of the substantia nigra indicating that 2D6-mediated detoxification will occur in target cells. 5. These data present evidence that CYP2D6 will be a factor in susceptibility to MPTP neuronal toxicity and provide a biochemical rationale for the genetic observations linking a polymorphism at the CYP2D6 locus with susceptibility to Parkinson's.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1997

Effects of single-residue substitutions on negative cooperativity in ligand binding to dihydrofolate reductase.

The effects of six amino acid substitutions in Lactobacillus casei dihydrofolate reductase, predominantly in the coenzyme binding site, on catalysis and on the negative cooperativity between NADPH and tetrahydrofolate binding have been determined. Replacement of Leu62, His64 or Leu54 by alanine has no effect on kcat, and produces only modest changes in negative cooperativity. Alanine substitution of His77, which interacts indirectly with the coenzyme adenine ring, leads to a doubling of the negative cooperativity and a consequent doubling of kcat. Replacement of Arg43, which interacts with the coenzyme 2'-phosphate, by alanine, or of Trp21, which interacts with the coenzyme nicotinamide ring, by histidine leads to a 20-100-fold decrease in negative cooperativity. In both mutants there is a decrease in kcat; isotope effects show that product release is largely rate-limiting in R43A, whereas in W21H hydride ion transfer is rate-limiting. 1H NMR has been used to obtain information on the extent of the structural changes produced by the substitutions. This varies from very local effects in H64A to very widespread effects in W21H. These changes are used as the basis for discussion of the mechanisms of the functional effects of the various substitutions. It is suggested that residues in helix C, beta-strand 3 and the beta3-beta4 loop may be involved in the transmission of effects between the coenzyme and substrate binding sites.

Binding Sites↗
PubMed1997

Role of transmembrane pH gradient and membrane binding in nisin pore formation.

Nisin is a cationic antimicrobial peptide that belongs to the group of lantibiotics. It is thought to form oligomeric pores in the target membrane by a mechanism that requires the transmembrane electrical potential delta psi and that involves local pertubation of the lipid bilayer structure. Here we show that nisin does not form exclusively voltage-dependent pores: even in the absence of a delta psi, nisin is able to dissipate the transmembrane pH gradient (delta pH) in sensitive Lactococcus lactis cells and proteoliposomes. The rate of dissipation increases with the magnitude of the delta pH. Nisin forms pores only when the delta pH is inside alkaline. The efficiency of delta psi-induced pore formation is strongly affected by the external pH, whereas delta pH-induced pore formation is rather insensitive to the external pH. Nisin(1-12), an amino-terminal fragment of nisin, and (des-deltaAla5)-(nisin(1-32) amide have a strongly reduced capacity to dissipate the delta psi and delta pH in cytochrome c oxidase proteoliposomes and L. lactis cells. Both variants bind with reduced efficiency to liposomes containing negatively charged phospholipids, suggesting that both ring A and rings C to E play a role in membrane binding. Nisin(1-12) competes with nisin for membrane binding and antagonizes pore formation. These findings are consistent with the wedge model of nisin-induced pore formation.

Animals↗
PubMed1996

Smooth muscle alternative splicing induced in fibroblasts by heterologous expression of a regulatory gene.

Alternative splicing is a common mechanism for regulating gene expression in different cell types. In order to understand this important process, the trans-acting factors that enforce the choice of particular splicing pathways in different environments must be identified. We have used the rat alpha-tropomyosin gene as a model system of tissue-specific alternative splicing. Exon 3 of alpha-tropomyosin is specifically inhibited in smooth muscle cells allowing the alternative inclusion of exon 2. We have used a novel gene transfer and selection strategy to detect a gene whose expression in fibroblasts is sufficient to switch them to smooth muscle-specific splicing of alpha-tropomyosin and also alpha-actinin. Extracts from the regulating fibroblasts contain an apparently novel 55 kDa protein which binds to RNA elements required for regulation of tropomyosin splicing. This protein is not detected in extracts of non-regulating cells and is therefore a strong candidate cell-specific splicing regulator. These experiments advance our understanding of smooth muscle splicing regulation as well as establishing a means for direct cloning of tissue-specific splicing regulators which have so far been refractory to biochemical analysis.

Actinin↗
PubMed1996

The conformation of coenzyme A bound to chloramphenicol acetyltransferase determined by transferred NOE experiments.

The conformation of coenzyme A bound to chloramphenicol acetyltransferase has been studied in solution by NMR methods. Transferred nuclear Overhauser enhancement (NOE) and rotating frame NOE (ROE) experiments were used to determine the conformation of the bound coenzyme. Experiments were carried out at five mixing times and two temperatures, and with normal and perdeuterated enzyme, to ensure (1) that the fast exchange condition was satisfied and (2) that the results were not complicated by spin diffusion involving enzyme protons. The data were analysed using a general approach involving combined exchange and relaxation matrices. For the binary complex of coenzyme A (CoA) and enzyme, the conformation of CoA was calculated by using distance constraints derived from the intensities of 71 NOE and 33 ROE cross-peaks between coenzyme protons. The conformation of the adenosine moiety of CoA in the structure deduced by NMR is very close to that seen in the crystal structure of this complex, while the pantetheine moiety is clearly less extended. Essentially the same conformation was obtained whether or not the calculations included the protein (with appropriate intermolecular energy terms). The difference between the NMR and X-ray structures is interpreted in terms of the existence of two conformations of the CoA-enzyme complex. Support for this model comes from measurements of the coenzyme dissociation rate constant; NMR (lineshape analysis and transferred NOE experiments) gives estimates of koff approximately 3700 s-1 at 298 K and approximately 500 s-1 at 280 K, both significantly greater than estimates by fluorescence stopped-flow measurements. For the ternary complex of CoA, chloramphenicol and enzyme, 71 NOE cross peaks between protons of coenzyme A and a further ten cross-peaks between protons of coenzyme A and chloramphenicol were measured. Starting with a model derived from the crystal structures of the two binary complexes (in the absence of crystallographic data for the ternary complex) the conformations and relative positions of the two ligands were refined using the distance constraints derived from these NOEs. The conformation of the adenosine part of CoA is the same as in the binary complex, while the pantetheine arm is more extended and approaches close to the bound chloramphenicol molecule. The model of the ternary complex is discussed in terms of the information available on the mechanism of the enzyme.

Chloramphenicol O-Acetyltransferase↗
PubMed1996

The solution structure of the bovine S100B protein dimer in the calcium-free state.

BACKGROUND: S100B (S100beta) is a member of the S100 family of small calcium-binding proteins: members of this family contain two helix-loop-helix calcium-binding motifs and interact with a wide range of proteins involved mainly in the cytoskeleton and cell proliferation. S100B is a neurite-extension factor and levels of S100B are elevated in the brains of patients with Alzheimer's disease or Down's syndrome: the pattern of S100B overexpression in Alzheimer's disease correlates with the pattern of neuritic-plaque formation. Identification of a growing class of S100 proteins and the likely neurochemical importance of S100B make the determination of the structure of S100B of interest. RESULTS: We have used NMR to determine the structure of the reduced S100B homodimer in the absence of calcium. Each monomer consists of a four-helix bundle, arranged in the dimer in an antiparallel fashion. The fourth helix of each monomer runs close to the equivalent helix of the other monomer for almost its full length, extending the hydrophobic core through the interface. The N-terminal, but not the C-terminal, calcium-binding loop is similar to the equivalent loop in the monomeric S100 protein calbindin and is in a conformation ready to bind calcium. CONCLUSIONS: The novel dimer structure reported previously for calcyclin (S100A6) is the common fold for the dimeric S100B proteins. Calcium binding to the C-terminal calcium-binding loop would be expected to require a conformational change, which might provide a signal for activation. The structure suggests regions of the molecule likely to be involved in interactions with effector molecules.

Amino Acid Sequence↗
PubMed1996

Structure-activity relationships in the peptide antibiotic nisin: antibacterial activity of fragments of nisin.

The post-translationally modified peptide antibiotic nisin has been cleaved by a number of proteases and the fragments produced purified, characterised chemically, and assayed for activity in inhibiting the growth of Lactococcus lactis MG1614 and Micrococcus luteus NCDO8166. These results provide information on the importance of different parts of the nisin molecule for its growth-inhibition activity. Removal of the C-terminal five residues leads to approximately a 10-fold decrease in potency, while removal of a further nine residues, encompassing two of the lanthionine rings, leads to a 100-fold decrease. There are some differences between analogous fragments of nisin and subtilin, suggesting possible subtle differences in mode of action. Cleavage within, or removal of, lanthionine ring C essentially abolishes the activity of nisin. The fragment nisin1-12 is inactive itself, and specifically antagonises the growth-inhibitory action of nisin. These results are discussed in terms of current models for the mechanism of action of nisin.

Amino Acid Sequence↗
PubMed1996

A model for human cytochrome P450 2D6 based on homology modeling and NMR studies of substrate binding.

The cytochrome P450 responsible for the debrisoquine/sparteine polymorphism (P450 2D6) has been produced in large quantities by expression of a modified cDNA in baculovirus. A polyhistidine extension was incorporated at the C-terminus of the expressed protein, which, after purification of the protein on a nickel-agarose column, could be removed proteolytically by treatment with thrombin. Purified yields of P450 2D6 were 2.4 mg from 700 mL of cell culture. The protein had a greater than 90% heme content and was fully active, having no residual absorbance at 420 nm in the reduced CO complex. The quantities produced allowed direct study of the interaction of the substrate codeine with the enzyme by paramagnetic relaxation effects on the NMR spectrum of the substrate. Distances between the heme iron atom and substrate protons were calculated from these experiments, and the orientation of the substrate in the binding pocket was determined. This showed that codeine was bound with the methoxy group of the molecule closest to the heme iron (iron-methyl proton distance of 3.1 +/- 0.1 A), consistent with the observed O-demethylation to morphine. A model of the complex Of P450 2D6 with codeine was built from a multiple sequence and structure alignment of the known crystal structures for P450s, incorporating the experimental constraints derived from the NMR studies. This showed that the overall fold Of P450 2D6 is more similar to that of P450 BM3 than to either P450 cam or P450 terp. Codeine binds to P450 2D6 so that the methoxy group is directly above the A ring of the heme, while the basic nitrogen interacts with the carboxylate of aspartate 301.

Amino Acid Sequence↗
PubMed1996

Functional high level expression of cytochrome P450 CYP2D6 using baculoviral expression systems.

Cytochrome P-450 CYP2D6 plays a central role in the metabolism of many widely used therapeutic drugs including beta-adrenergic antagonists, antiarrhythmics, and tricyclic antidepressants. Recombinant baculoviruses have been constructed containing the full-length human CYP2D6 cDNA and used to express CYP2D6 in Spodoptera frugiperda (Sf9) cells. High levels of recombinant protein have been produced using either polyhedrin or basic protein promoters (0.05-0.20 nmol/mg cell protein; 0.05-0.15 nmol/liter). The enzyme is catalytically active toward CYP2D6 substrates such as bufuralol and metoprolol. In order to optimize catalytic activity human reductase was coexpressed with CYP2D6 in Sf9 cells; reductase activity was in the region of 1000-1500 units per mg cell protein, while spectrally active CY2D6 was in the range 10-20 pmol/mg cell protein. The K(m) and K(cat) values for bufuralol metabolism were estimated as 4.7 microM and 12.23 min-1, respectively. The use of the conventional very late promoters such as the polyhedrin promoter generate a large proportion of inactive CYP2D6. The problem was to a degree circumvented using the "late" basic promoter which is active earlier in the baculovirus infection cycle. The yield of functional CYP2D6 was at least as high as with very late promoters, but the proportion of inactive protein was reduced. Bufuralol hydroxylase activity could be measured directly by HPLC analysis of cell culture media supplemented with bufuralol, and we have developed a plate assay system which provides a simple method for the analysis of drug metabolism reactions using Sf9 cells. Expression using baculovirus provides a valuable source of functional CYP2D6 for work aimed at elucidating the structure and function of the enzyme.

Adrenergic beta-Antagonists↗
PubMed1996

The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme.

Beta-Lactamases are widespread in the bacterial world, where they are responsible for resistance to penicillins, cephalosporins, and related compounds, currently the most widely used antibacterial agents. Detailed structural and mechanistic understanding of these enzymes can be expected to guide the design of new antibacterial compounds resistant to their action. A number of high-resolution structures are available for class A beta-lactamases, whose catalytic mechanism involves the acylation of a serine residue at the active site. The identity of the general base which participates in the activation of this serine residue during catalysis has been the subject of controversy, both a lysine residue and a glutamic acid residue having been proposed as candidates for this role. We have used the pH dependence of chemical modification of epsilon-amino groups by 2,4,6,-trinitrobenzenesulfonate and the pH dependence of the epsilon-methylene 1H and 13C chemical shifts (in enzyme selectively labeled with [epsilon-13C]lysine) to estimate the pKa of the relevant lysine residue, lysine-73, of TEM-1 beta-lactamase. Both methods show that the pKa of this residue is > 10, making it very unlikely that this residue could act as a proton acceptor in catalysis. An alternative mechanism in which this role is performed by glutamate-166 through an intervening water molecule is described.

Binding Sites↗
PubMed1996

NMR studies of the mode of binding of corepressors and inducers to Escherichia coli trp repressor.

The binding of the corepressors tryptophan and 5-methyltryptophan and of the inducers 3-indolepropionate, 3-indoleacrylate and 5-methylindole to the Escherichia coli trp repressor have been studied by 1H-NMR spectroscopy. Identification of the resonances of the protons of bound ligands and their NOEs to protons of the protein (measured as transferred NOE) was greatly facilitated by the use of samples of the protein in which the hydrogens of all residues except alanine, isoleucine and threonine was replaced by deuterium. Chemical-shift changes of protein-backbone resonances and side-chain-amide resonances on ligand binding were measured with generally or selectively 15N-labelled protein. The patterns of changes in the chemical shifts of protein resonances and, particularly, ligand resonances distinguish the corepressors from the inducers, indicating, in agreement with earlier work, that corepressors and inducers bind to the protein in different ways. The NOEs observed for the bond ligands have been used to determine the position of the ligands in the crystallographically determined binding site, by means of a simulated-annealing molecular-dynamics protocol. The structures obtained show that the orientation in the binding site of the indole rings of tryptophan and 5-methyltryptophan and of 3-indolepropionate and 3-indoleacrylate differ by approximately 180 degrees in solution (in agreement with the crystallographic data for complexes of the trp repressor with tryptophan or with 3-indolepropionate). The value and limitations of calculating ligand positions based on transferred NOE are discussed.

Bacterial Proteins↗
PubMed1996

Mechanism of lantibiotic-induced pore-formation.

Nisin and other lantibiotics have a bacteriocidal effect against Gram-positive bacteria, and also inhibit the outgrowth of bacterial spores. The bacteriocidal effect appears to be due to the formation of pores in the bacterial membrane. In the absence of anionic membrane phospholipids, the lantibiotic nisin acts as an anion selective carrier. In the presence of anionic phospholipids, nisin forms nonselective, transient, multi-state pores in cells, proteoliposomes, liposomes and black lipid membranes. Pore formation involves distinct steps. First, nisin associates tightly with the anionic membrane surface leading to a high local concentration. This results in a disturbance of the lipid dynamics near the phospholipid polar head group-water interface, and an immobilization of lipids. In the presence of a transmembrane electrical potential above the threshold level, the molecules reorient, presumably as an aggregate, from a surface-bound into a membrane-inserted configuration. Co-insertion of bound, anionic phospholipids results in bending of the lipid surface giving rise to a wedge-like, nonspecific, water-filled pore.

Amino Acid Sequence↗
PubMed1996

The other kind of biological NMR--studies of enzyme-substrate interactions.

NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in drug metabolism. Cytochromes P450 play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate complexes. The other two enzymes, chloramphenicol acetyltransferase and beta-lactamase, are responsible for bacterial resistance to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme A bound to the enzyme, while in the case of beta-lactamase the pK of a specific lysine residue at the active site has been determined, providing valuable information on the catalytic mechanism.

Binding Sites↗