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R P Bywater

Publications and source records attributed to R P Bywater.

At least 19 recordsLinked to original sources
PubMed2002

Construction of the simplest model to explain complex receptor activation kinetics.

We study the mathematical solutions to the kinetic equations arising from various simple ligand-receptor [corrected] models. Focusing on the prediction of the various models for the activity vs. concentration curve, we find that solutions to the kinetic equations arising from the so-called dimer model exhibit features observed in some experiments, most noticeably a distinct maximum in the activity curve.

Animals↗
PubMed2001

Dimerization of G-protein-coupled receptors.

The evolutionary trace (ET) method, a data mining approach for determining significant levels of amino acid conservation, has been applied to over 700 aligned G-protein-coupled receptor (GPCR) sequences. The method predicted the occurrence of functionally important clusters of residues on the external faces of helices 5 and 6 for each family or subfamily of receptors; similar clusters were observed on helices 2 and 3. The probability that these clusters are not random was determined using Monte Carlo techniques. The cluster on helices 5 and 6 is consistent with both 5,6-contact and 5,6-domain swapped dimer formation; the possible equivalence of these two types of dimer is discussed because this relates to activation by homo- and heterodimers. The observation of a functionally important cluster of residues on helices 2 and 3 is novel, and some possible interpretations are given, including heterodimerization and oligomerization. The application of the evolutionary trace method to 113 aligned G-protein sequences resulted in the identification of two functional sites. One large, well-defined site is clearly identified with adenyl cyclase, beta/gamma and regulator of G-protein signaling (RGS) binding. The other G-protein functional site, which extends from the ras-like domain onto the helical domain, has the correct size and electrostatic properties for GPCR dimer binding. The implications of these results are discussed in terms of the conformational changes required in the G-protein for activation by a receptor dimer. Further, the implications of GPCR dimerization for medicinal chemistry are discussed in the context of these ET results.

Amino Acid Sequence↗
PubMed2001

Conformation of alamethicin in oriented phospholipid bilayers determined by (15)N solid-state nuclear magnetic resonance.

The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.

Alamethicin↗
PubMed2001

Influence of a lipid interface on protein dynamics in a fungal lipase.

Lipases catalyze lipolytic reactions and for optimal activity they require a lipid interface. To study the effect of a lipid aggregate on the behavior of the enzyme at the interfacial plane and how the aggregate influences an attached substrate or product molecule in time and space, we have performed molecular dynamics simulations. The simulations were performed over 1 to 2 ns using explicit SPC water. The interaction energies between protein and lipid are mainly due to van der Waals contributions reflecting the hydrophobic nature of the lipid molecules. Estimations of the protonation state of titratable residues indicated that the negative charge on the fatty acid is stabilized by interactions with the titratable residues Tyr-28, His-143, and His-257. In the presence of a lipid patch, the active site lid opens wider than observed in the corresponding simulations in an aqueous environment. In that lid conformation, the hydrophobic residues Ile-85, Ile-89, and Leu-92 are embedded in the lipid patch. The behavior of the substrate or product molecule is sensitive to the environment. Entering and leaving of substrate molecules could be observed in presence of the lipid patch, whereas the product forms strong hydrogen bonds with Ser-82, Ser-144, and Trp-88, suggesting that the formation of hydrogen bonds may be an important contribution to the mechanism by which product inhibition might take place.

Binding Sites↗
PubMed2001

A sequence and structural study of transmembrane helices.

A comparison is made between the distribution of residue preferences, three dimensional nearest neighbour contacts, preferred rotamers, helix-helix crossover angles and peptide bond angles in three sets of proteins: a non-redundant set of accurately determined globular protein structures, a set of four-helix bundle structures and a set of membrane protein structures. Residue preferences for the latter two sets may reflect overall helix stabilising propensities but may also highlight differences arising out of the contrasting nature of the solvent environments in these two cases. The results bear out the expectation that there may be differences between residue type preferences in membrane proteins and in water soluble globular proteins. For example, the beta-branched residue types valine and isoleucine are considerably more frequently encountered in membrane helices. Likewise, glycine and proline. residue types normally associated with 'helix-breaking' propensity are found to be relatively more common in membrane helices. Three dimensional nearest neighbour contacts along the helix, preferred rotamers, and peptide bond angles are very similar in the three sets of proteins as far as can be ascertained within the limits of the relatively low resolution of the membrane proteins dataset. Crossing angles for helices in the membrane protein set resemble the four helix bundle set more than the general non-redundant set, but in contrast to both sets they have smaller crossing angles consistent with the dual requirements for the helices to form a compact structure while having to span the membrane. In addition to the pairwise packing of helices we investigate their global packing and consider the question of helix supercoiling in helix bundle proteins.

Amino Acid Sequence↗
PubMed2001

Dynamics of the substrate binding pocket in the presence of an inhibitor covalently attached to a fungal lipase.

To gain insight into the mobility of the occupied ligand-binding pocket of the Rhizomucor miehei lipase we have conducted a rigorous molecular dynamics analysis. The covalently attached inhibitor, ethylhexylphosphonate, was employed as a mimic of the putative tetrahedral intermediate in the esterolytic reaction. Our results show that in this lipase, ligand recognition is influenced by the flexibility of the binding pocket, a feature that is common to many other enzymes. Several regions around the active site were found to move significantly to adapt to the inhibitor. These motions are correlated to the flexibility of the inhibitor. In particular, the hexyl chain of the inhibitor shows considerable mobility, and adjacent residues in the binding cleft accommodate to this flexibility. Pronounced fluctuations in the binding pocket induced by the flexibility of the inhibitor are observed in the hinge region F79-S82, the active site loop region W88-V95 and the protein regions P209-F215/H257-Y260. The flexibility in the regions F79-S82 and H257-Y260, where the shorter ethyl chain is located, indicates that additional space in this binding cleft region is available for accommodating a larger moiety. Fluctuations in the region W88-V95 and P209-F215 are due to the relatively short flexible hexyl carbon chain. This part of the binding pocket could be stiffened by the presence of a longer carbon chain. Though the inhibitor is covalently attached through the phosphonate moiety, interaction of the remainder of the molecule and the enzyme are determined by hydrophobic interactions, where the Van der Waals energies are approximately 25% lower than the electrostatic contributions.

Catalytic Domain↗
PubMed2001

Lipid-facing correlated mutations and dimerization in G-protein coupled receptors.

A correlated mutation analysis has been performed on the aligned protein sequences of a number of class A G-protein coupled receptor families, including the chemokine, neurokinin, opioid, somatostatin, thyrotrophin and the whole biogenic amine family. Many of the correlated mutations are observed flanking or neighbouring conserved residues. The correlated residues have been plotted onto the transmembrane portion of the rhodopsin crystal structure. The structure shows that a significant proportion of the correlated mutations are located on the external (lipid-facing) region of the helices. The occurrence of these highly correlated patterns of change amongst the external residues suggest that they are sites for protein-protein interactions. In particular, it is suggested that the correlated residues may be involved in either large conformational changes, the formation of heterodimers or homodimers (which may be domain swapped) or oligomers required for activation or internalization. The results are discussed in the light of the subtype-specific heterodimerization observed for the chemokine, opioid and somatostatin receptors.

Amino Acid Sequence↗
PubMed2000

Essential motions and energetic contributions of individual residues in a peptide bound to an SH3 domain.

We have studied protein-ligand interactions by molecular dynamics simulations using software designed to exploit parallel computing architectures. The trajectories were analyzed to extract the essential motions and to estimate the individual contributions of fragments of the ligand to overall binding enthalpy. Two forms of the bound ligand are compared, one with the termini blocked by covalent derivatization, and one in the underivatized, zwitterionic form. The ends of the peptide tend to bind more loosely in the capped form. We can observe significant motions in the bound ligand and distinguish between motions of the peptide backbone and of the side chains. This could be useful in designing ligands, which fit optimally to the binding protein. We show that it is possible to determine the different contributions of each residue in a peptide to the enthalpy of binding. Proline is a major net contributor to binding enthalpy, in keeping with the known propensity for this family of proteins to bind proline-rich peptides.

Amino Acid Sequence↗
PubMed2000

Structure of Fab hGR-2 F6, a competitive antagonist of the glucagon receptor.

The monoclonal antibody hGR-2 F6 has been raised against the human glucagon receptor and shown to act as a competitive antagonist. As a first step in the structural characterization of the receptor, the crystal structure of the Fab fragment from this antibody is reported at 2.1 A resolution. The hGR-2 F6 Fab crystallizes in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 76.14, b = 133.74, c = 37.46 A. A model generated by homology modelling was used as an aid in the chain-tracing and the Fab fragment structure was subsequently refined (final R factor = 21.7%). The structure obtained exhibits the typical immunoglobulin fold. Complementarity-determining regions (CDRs) L1, L2, L3, H1 and H2 could be superposed onto standard canonical CDR loops. The H3 loop could be classified according to recently published rules regarding loop length, sequence and conformation. This loop is 14 residues long, with an approximate beta-hairpin geometry, which is distorted somewhat by the presence of two trans proline residues at the beginning of the loop. It is expected that this H3 loop will facilitate the design of synthetic probes for the glucagon receptor that may be used to investigate receptor activity.

Amino Acid Sequence↗
PubMed2000

Structure-function analysis of human CYP3A4 using a specific proinhibitory antipeptide antibody.

An anti-peptide antibody targeted against residues 253 to 269 of human CYP3A4 was produced that specifically and potently inhibited its activity in human hepatic microsomal fraction (>90%). The function of this region in P450 catalysis was investigated. Antibody binding to CYP3A4 was unable to affect the magnitude of the Type I spectrum on addition of testosterone. It also had no effect on the K(m) of the enzyme for testosterone, but it did cause a marked decrease in V(max) (>90%) of testosterone 6 beta-hydroxylation. There was no change in the ability of the antibody-bound CYP3A4 to form the steady-state level of the enzymatically or chemically reduced P450-CO complex or even the steady-state level of the dioxy-ferrous complex during testosterone metabolism, but the oxidation of NADPH by CYP3A4 in the presence of antibody was 60% that of CYP3A4 in the absence of antibody. The binding of the antibody also resulted in potent inhibition of cumene hydroperoxide-supported testosterone 6 beta-hydroxylase activity of human liver microsomal fraction (>90%). Our conclusion is that the loop region targeted in CYP3A4 is not involved in substrate binding, in reductase binding, in the transfer of the first or second electron from the reductase to CYP3A4, or in the binding of molecular oxygen. We speculate that antibody binding to CYP3A4 inhibits enzyme activity by destabilizing the ternary hydroperoxo complex, by interfering with the second proton transfer, and/or by interfering with the conformational changes that are suggested to be induced by substrate binding.

Amino Acid Sequence↗
PubMed1999

Structure of the integral membrane domain of the GLP1 receptor.

A three-dimensional (3D) model of the integral membrane domain of the GLP1 receptor, a member of the secretin receptor family of the G-protein-coupled receptor superfamily is proposed. The probable arrangement of the seven helices in this receptor was deduced from a detailed analysis of all the sequences in the secretin receptor family. The analysis includes: 1) identifying the transmembrane helices, 2) charge distribution analysis to estimate to which extent the transmembrane helices are buried, 3) Fourier transform analysis of different property profiles within the transmembrane helices to determine the orientation of exposed and buried faces of the helices, 4) alignment of sequences with those of the rhodopsin-like family using the novel "cold spot" method reported herein, 5) determination of lengths of transmembrane helices and their connecting loops and the constraints these impose on packing, tilting and organization, 6) incorporation of mutagenesis and ligand specificity data. We find that there is a close similarity between the structural properties of receptors of the secretin family and those of the rhodopsin-like family as typified by the frog rhodopsin structure recently solved by electron cryomicroscopy.

Amino Acid Sequence↗
PubMed1999

Functional concerted motions in the bovine serum retinol-binding protein.

The large concerted motions in the apo/holo bovine serum retinol-binding protein were studied using molecular dynamics simulation and 'essential dynamics' analysis. Initially, concerted motions were calculated from conformational differences between various crystal structures. The dynamic behaviour of the protein in the configurational space directions, described by these concerted motions, is analysed. This reveals that the large backbone dynamics of the protein is not influenced by the presence of retinol. Study of free retinol dynamics and retinol in the retinol binding site reveals that the protein binds retinol in a favourable conformation, as opposed to what has been previously described for the bovine cellular retinol-binding protein.

Animals↗
PubMed1999

Computational analysis of chain flexibility and fluctuations in Rhizomucor miehei lipase.

We have performed molecular dynamics simulation of Rhizomucor miehei lipase (Rml) with explicit water molecules present. The simulation was carried out in periodic boundary conditions and conducted for 1. 2 ns in order to determine the concerted protein dynamics and to examine how well the essential motions are preserved along the trajectory. Protein motions are extracted by means of the essential dynamics analysis method for different lengths of the trajectory. Motions described by eigenvector 1 converge after approximately 200 ps and only small changes are observed with increasing simulation time. Protein dynamics along eigenvectors with larger indices, however, change with simulation time and generally, with increasing eigenvector index, longer simulation times are required for observing similar protein motions (along a particular eigenvector). Several regions in the protein show relatively large fluctuations and in particular motions in the active site lid and the segments Thr57-Asn63 and the active site hinge region Pro101-Gly104 are seen along several eigenvectors. These motions are generally associated with glycine residues, while no direct correlations are observed between these fluctuations and the positioning of prolines in the protein structure. The partial opening/closing of the lid is an example of induced fit mechanisms seen in other enzymes and could be a general mechanism for the activation of Rml.

Amino Acid Sequence↗
PubMed1998

Domain swapping in G-protein coupled receptor dimers.

Computer simulations were performed on models of the beta2-adrenergic receptor dimer, including 5,6-domain swapped dimers which have been proposed as the active, high affinity form (here the dimer interface lies between helices 5 and 6). The calculations suggest that the domain swapped dimer is a high energy structure in both the apo dimer and in the presence of propranolol. In the presence of agonist the energy of the domain swapped dimer is significantly lowered. Analysis of the dimer structure suggests that the agonist-induced conformational change optimizes the helix-helix interactions at the 5-6 interface. An antagonist on the other hand has little effect on these interactions. These observations are consistent with the hypothesis that the agonist functions by shifting the equilibrium in favour of the domain swapped dimer. Indirect support for the domain swapping hypothesis was obtained from the correlated mutations amongst the external residues of the known beta2-adrenergic receptors. These occur mainly at the 5-6 interface at precisely the locations predicted by the simulations; site-directed mutagenesis data in support of a functional role for these lipid-facing correlated residues is presented. The article includes a review of the experimental evidence for G-protein coupled receptor dimerization. Many other aspects of G-protein coupled receptor activation are discussed in terms of this domain swapping hypothesis

Amino Acid Sequence↗
PubMed1993

PLIM: a protein-ligand interaction modeller.

A computer program is presented that models the binding of selected chemical groups to a protein surface. The groups are successively incorporated at energetically favourable positions to build up a pharmacophore pattern that may be used as the basis for a database search for possible ligands. The ability to predict known binding points in a trypsin-inhibitor complex is demonstrated, and the results from a run on dihydrofolate reductase are shown to be usable as a pharmacophore pattern for a database search.

Animals↗
PubMed1983

Particle geometry and the chromatographic separation of cells: the simplest case of uniform particles.

A theoretical study, using numerical methods, of the problem of packing spherical particles has been carried out in order to design an optimal chromatographic packing material for cell separation. It is concluded that a monodisperse packing of hard beads does not yield a packing in which narrow crevices, impenetrable to cells, are excluded. Allowing a certain degree of compressibility alleviates this problem somewhat.

Cell Separation↗