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J Warwicker

Publications and source records attributed to J Warwicker.

32 records · Page 2Linked to original sources
PubMed1995

A molecular model for the redox potential difference between thioredoxin and DsbA, based on electrostatics calculations.

The disulphide active sites of thioredoxin and DsbA are known to possess a high degree of structural homology. However, DsbA is a much stronger oxidant than thioredoxin. The redox potential difference between DsbA and thioredoxin has been measured to be 160 mV, equivalent to a shift of 15.4 kJ/mol in the reduced/oxidised equilibrium. Electrostatics calculations have been used to study the relative stabilities of the reduced forms of the two proteins. Model calculations suggest that much of the redox potential difference between DsbA and thioredoxin arises form altered stabilisation of the exposed and ionised thiolates of the reduced forms, supporting suggestions previously made on the basis of experimental studies. The calculations have been used to construct a molecular model for the difference in thiolate stabilisation. Although specific interactions, such as thiolate-NH 35 (thioredoxin)/33 (DsbA), provide substantial stabilisation in each reduced protein, the difference between thioredoxin and DsbA is predicted to reside in several side-chain and main-chain groups acting in concert. Residues H32 and Q97 in DsbA are predicted to contribute, along with substantial regions of the polypeptide backbone in the protein domain which is common to DsbA and thioredoxin. Increased thiolate stabilisation by the peptide dipoles is suggested to arise from altered main-chain disposition, and the effect of the additional protein domain of DsbA on the electric field. Peptide dipoles in a region of about 20 residues close to the active site disulphide are predicted to contribute significantly to the redox potential difference.

Binding Sites↗
PubMed1995

A model for vicilin solubility at mild acidic pH, based on homology modelling and electrostatics calculations.

The crystallographic structures of jack bean canavalin and French bean phaseolin have been used to construct a homology model of the storage vicilin of cocoa. Reported molecular weights for cocoa storage protein subunits correlate with proteolysis at the site of a large hydrophilic insert in the mature protein. Burial of the hydrophobic amino acids on trimer formation is a strongly conserved feature in the vicilin family. Histidine residues also sit at the monomer-monomer interfaces of the trimer and are likely to contribute to the decreased solubility of cocoa vicilin at mild acidic pH, which is generally considered to be caused solely by aggregation near to the isoelectric point. Electrostatic calculations suggest that such an arrangement of histidine residues in the absence of specific counterion binding will not favour the particular geometry of trimer formation below neutral pH. Higher order aggregates that do not exclude histidine charge from the solvent may be favoured, aiding the precipitation of cocoa vicilin at mild acidic pH. This suggestion is considered for the vicilin family. The hypothesis could contribute to an understanding of the pH and ionic strength dependence of vicilin solubility in vitro, and possibly of the behaviour of vicilins in the seed storage environment.

Amino Acid Sequence↗
PubMed1994

Improved continuum electrostatic modelling in proteins, with comparison to experiment.

Electrostatic interactions in macromolecules can be calculated with the method of finite differences applied to a continuum model. The accuracy of dielectric and counterion continuum modelling has been tested for long-range interactions by comparison with available experimental data over a range of ionic strengths. Various model parameters have been adjusted. Some have little effect, such as protein dielectric and the selection of Van der Waals radii. It is shown that the reduction in interaction due to dielectric effects is overestimated when a dielectric constant of 80 is assigned to all solvent accessible regions. Improved agreement is seen when the effects of the Kirkwood correlation sphere and dielectric saturation are included. Further support for the use of dielectric saturation arises from a correlation of solvent polarization saturation with crystallographic ordered water structure. Calculations over the medium ionic strength range indicate that requiring counterions to maintain a solvent layer places too great a restriction on their approach to the protein-solvent interface. However, counterion accessibility that coincides with the solvent accessible region gives too much interaction damping. Modelling of observed ion binding sites suggests that a counterion response which includes an ion desolvation term, obtained by difference calculation, will improve the computation of ionic strength effects. This study demonstrates that there is scope for improvement in continuum electrostatics calculations, and shows that progress is possible with the inclusion of physically realistic solvent and counterion properties at the protein surface.

Amino Acid Sequence↗
PubMed1994

The activity of porcine pancreatic phospholipase A2 in 20% alcohol/aqueous solvent, by experiment and electrostatics calculations.

The activity of porcine pancreatic phospholipase A2 (pla2), measured at pH 8, is reduced when methanol or ethanol is added to the aqueous solution. Finite difference electrostatics calculations were used to study the effect of modelling mixed solvents on the pKas of histidine 48 and the amino-terminal group, both of which influence the pH-dependence of catalysis. Calculations and experiment indicate that these pKa values cannot account for the activity reduction. Charge separation in the transition state is destabilized in 20% alcohol solvent relative to 100% aqueous solvent. The calculated values, which are combinations of stabilizing and destabilizing contributions, are in qualitative agreement with experiment. Saturating dielectric theory is used to model solvent water ordering in a high electric field, and water dielectric structure is assumed to dominate at the 20% alcohol level. The observed agreement demonstrates the utility of transition state stabilization theory and continuum solvent modelling. It is further suggested that electrostatic effects on kcat contribute to the pH-dependence of activity around pH 7, and to previously reported activity changes for charge mutants.

Amino Acid Sequence↗
PubMed1994

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions.

The role of electrostatic interactions between the ionizable Asp158 and the active site thiolate-imidazolium ion pair of some cysteine proteinases has been the subject of controversy for some time. This study reports the expression of wild type procaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants from Escherichia coli. Purification of autocatalytically matured enzymes yielded sufficient fully active material for pH (kcat/Km) profiles to be obtained. Use of both uncharged and charged substrates allowed the effects of different reactive enzyme species to be separated from the complications of electrostatic effects between enzyme and substrate. At least three ionizations are detectable in the acid limb of wild type caricain and the Glu and Asn mutants. Only two pKa values, however, are detectable in the acid limb using the Ala mutant. Comparison of pH activity profiles shows that whilst an ionizable residue at position 158 is not essential for the formation of the thiolate-imidazolium ion pair, it does form a substantial part of the electrostatic field responsible for increased catalytic competence. Changing the position of this ionizable group in any way reduces activity. Complete removal of the charged group reduces catalytic competence even further. This work indicates that hydronations distant to the active site are contributing to the electrostatic effects leading to multiple active ionization states of the enzyme.

Amino Acid Sequence↗
PubMed1992

Model for the differential stabilities of rhinovirus and poliovirus to mild acidic pH, based on electrostatics calculations.

Previous calculations of electrostatic interactions in the rhinovirus capsid have identified a subset of histidine residues, paired with lysine or arginine, that may be involved in pH-induced conformational changes related to viral uncoating. Further calculations with the finite difference method, accounting for the dielectric environment of the ionizable groups, suggest that charge burial in the crystal conformation will prevent protonation of these histidine residues in the pentamer-pentamer interface. Calculations with a modelled pentamer-pentamer interface in which three beta-strands are removed recover mildly acidic pKa values for the histidines. These results are discussed in the context of the structural interactions of these three beta-strands, which form a beta-sheet extension from the rest of the capsid, and with regard to the conformation of the homologous beta-sheet extension in poliovirus, which also possesses homologous histidine-lysine/arginine pairs. A model is developed in which the structural stability of the beta-sheet extension is related to the difference in acid stability of rhinovirus and poliovirus. It is suggested that, for poliovirus prior to cell receptor binding, the beta-sheet extension is stable at pH 3, the pentamer-pentamer interface histidines remain buried, and the virus is acid-stable. Cell receptor binding of poliovirus destabilizes the beta-sheet extension and the acid lability that is proposed to result could be involved in viral uncoating. For rhinovirus it is suggested that the observed conformational change in the absence of cell receptor binding involves a further acidic pH-activated process or conformational fluctuations that rearrange the beta-sheet extension and expose the pentamer-pentamer interface histidine residues to the acidic medium. Sequence analysis and electrostatics calculations reveal an aspartic acid in the beta-sheet extension that may have different pKa values in rhinovirus and poliovirus.

Amino Acid Sequence↗
PubMed1991

Modification of the stability of phospholipase A2 by charge engineering.

Electrostatic interactions play an important role in stabilizing the folded conformation of globular proteins. Here we predict the change in stability of charge engineered mutants, construct these mutants and compare the predicted change in stability with that observed. The change in stability was correctly predicted for two of the three mutants and the factors responsible for the discrepancy between observation and prediction for the third mutant are discussed.

Enzyme Stability↗
PubMed1991

Changes in activity of porcine phospholipase A2 brought about by charge engineering of a major structural element to alter stability.

We have modified the stability of porcine phospholipase A2 by charge engineering. The mutations are situated at the N-terminal of a major helix and are N89D and N89D/E92Q. This engineering has significantly altered the activity of the enzyme to aggregated and monomeric substrates. A N89D/E92K mutant is more stable but considerably less active than wild type. An N89D mutant is more stable and of similar activity to wild type. The substantial change in activity may be due to direct interaction of residue 92 with aggregated substrate or may be via second calcium binding. Second calcium binding may be more probable as activity against monomers is also affected. Additional calcium binding may therefore be an important way of manipulating the activity of phospholipase A2.

Animals↗
PubMed1989

A theoretical study of the acidification of the rhinovirus capsid.

Electrostatic calculations for human rhinovirus 14 indicate that histidine-base residue pairs in the region of a beta-strand interaction between pentamers may be involved in a pH-induced process that leads to the release of viral RNA. Other picornavirus sequences are examined for these residue pairs, a subset of which is present in enteroviruses. Foot and mouth disease virus possesses one of the residue pairs, and cardioviruses, which undergo a separate pH and halide ion-induced capsid dissociation, possess none.

Capsid↗
PubMed1989

Investigating protein-protein interaction surfaces using a reduced stereochemical and electrostatic model.

A method of calculating the electrostatic potential energy between two molecules, using finite difference potential, is presented. A reduced charge set is used so that the interaction energy can be calculated as the two static molecules explore their full six-dimensional configurational space. The energies are contoured over surfaces fixed to each molecule with an interactive computer graphics program. For two crystal structures (trypsin-trypsin inhibitor and anti-lysozyme Fab-lysozyme), it is found that the complex corresponds to highly favourable interacting regions in the contour plots. These matches arise from a small number of protruding basic residues interacting with enhanced negative potential in each case. The redox pair cytochrome c peroxidase-cytochrome c exhibits an extensive favourably interacting surface within which a possible electron transfer complex may be defined by an increased electrostatic complementarity, but a decreased electrostatic energy. A possible substrate transfer configuration for the glycolytic enzyme pair glyceraldehyde phosphate dehydrogenase-phosphoglycerate kinase is presented.

Animals↗
PubMed1987

Electrostatic calculations and model-building suggest that DNA bound to CAP is sharply bent.

Two observations suggest that DNA, upon binding to E. coli catabolite gene activator protein (CAP), is sharply bent by a total angle of at least 100-150 degrees: (1) The electrostatic potential field of CAP shows regions of positive potential that form a ramp on 3 sides of the protein. (2) The DNA binding site size as determined by DNA ethylation interference with binding, (Majors: "Control of the E. coli Lac Operon at the Molecular Level." Ph.D. Thesis, Harvard University, Cambridge, 1977) and by relative affinities of DNA fragments of various lengths (Liu-Johnson et al.: Cell 47:995-1005, 1986) requires severe bending of the DNA to maintain its favorable electrostatic contact with the protein.

Bacterial Proteins↗
PubMed1986

Continuum dielectric modelling of the protein-solvent system, and calculation of the long-range electrostatic field of the enzyme phosphoglycerate mutase.

The numerical continuum electrostatic method presented previously (Warwicker, J. & Watson, H. C. (1982) J. Mol. Biol., 157, 671-679), is developed with an improved analysis of the protein-solvent system. Inclusion in the model of saturable solvent dielectric, and counterions is discussed and presented. A number of long-range electrostatic field calculations are made on bovine pancreatic trypsin inhibitor to demonstrate the differences between various solvent and counterion models. The long-range potential field, due to polar side-chain and alpha-helix dipole charge, is calculated for the glycolytic enzyme phosphoglycerate mutase. The positive potential in and around the catalytic cleft region is sufficiently large to suggest that it may play a role in long-range attraction of the enzyme's negatively charged substrates. Analogous systems with charge-charge interactions in solvent water are considered. It is suggested that a long-range enzyme-substrate attractive force-field may, in part, offset the repulsive energy arising from overlap of hydration shells between enzyme and substrate.

Aprotinin↗
PubMed1985

Electrostatic field of the large fragment of Escherichia coli DNA polymerase I.

The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy.

DNA Polymerase I↗