Biomedical subjects

S L Mowbray

Publications and source records attributed to S L Mowbray.

At least 37 records · Page 2Linked to original sources

PubMed1995

C alpha-based torsion angles: a simple tool to analyze protein conformational changes.

A simple method is presented for the analysis of protein conformational changes based on the comparison of torsion angles defined by four consecutive C alpha atoms. The technique was applied successfully to proteins that undergo hinge motion and shear motion. In the case of both MBP and LAO, which represent examples of hinge motion, the plot of the differences in C alpha-torsion angles between the open and closed forms of the proteins helped us to formulate a more thorough description of the conformational change: a large displacement of one domain with respect to the other where one of the domains does not behave like a rigid body but exhibits some degree of flexibility. The analysis of citrate synthase, which is an example of shear motion, shows that the largest differences in C alpha-torsion angles between the open and closed conformations are clustered around residues that belong to segments connecting alpha-helices, whereas the helices themselves appear to be rigid; this is in agreement with previous results obtained by detailed least-squares superpositions (Lesk AM, Chothia C, 1984, J Mol Biol 174:175-191).

Calcium-Binding Proteins↗

PubMed1995

Crystal structure of the dipeptide binding protein from Escherichia coli involved in active transport and chemotaxis.

The Escherichia coli periplasmic dipeptide binding protein functions in both peptide transport and taxis toward peptides. The structure of the dipeptide binding protein in complex with Gly-Leu (glycyl-L-leucine) has been determined at 3.2 A resolution. The binding site for dipeptides is designed to recognize the ligand's backbone while providing space to accommodate a variety of side chains. Some repositioning of protein side chains lining the binding site must occur when the dipeptide's second residue is larger than leucine. The protein's fold is very similar to that of the Salmonella typhimurium oligopeptide binding protein, and a comparison of the structures reveals the structural basis for the dipeptide binding protein's preference for shorter peptides.

Bacterial Proteins↗

PubMed1995

Modeling of the structure of the Haemophilus influenzae heme-binding protein suggests a mode of heme interaction.

The structure and function of the periplasmic heme-binding protein HbpA of Haemophilus influenzae were investigated. This protein is involved in the import of heme into the bacteria through the inner membrane, and thus is a key element of the organism's ability to survive in blood. A high degree of sequence similarity between HbpA and the dipeptide-binding protein of Escherichia coli is suggested to be the result of a functional relationship. An HbpA model built using the dipeptide-binding protein suggests a mode of heme binding that is distinct from those known in proteins of the human host. These results provide a starting point for rational drug design.

Bacterial Proteins↗

PubMed1995

Bacterial chemotaxis: a field in motion.

Many different types of studies are being combined to provide an increasingly detailed picture of the bacterial chemotaxis system. The structures of periplasmic receptors and a cytoplasmic response regulator, along with structures of domains of a membrane receptor, a receptor-modifying enzyme and a cytoplasmic histidine kinase, have been determined. These structures provide a basis for other work which is likely to open up new structural avenues.

Bacterial Proteins↗

PubMed1994

Probing protein-protein interactions. The ribose-binding protein in bacterial transport and chemotaxis.

A number of mutations at Gly134 of the periplasmic ribose-binding protein of Escherichia coli were examined by a combined biochemical and structural approach. Different mutations gave rise to different patterns of effects on the chemotaxis and transport functions. The smallest residue (alanine) had the least effect on transport, whereas large hydrophobic residues had the smallest effect on chemotaxis. Comparison of the x-ray crystal structure of the G134R mutant protein (2.5-A resolution) to that of the wild type (1.6-A resolution) showed that the basic structure of the protein was unaltered. The loss of chemotaxis and transport functions in this and similar mutant proteins must therefore be caused by relatively simple surface effects, which include a change in local main chain conformation. The loss of chemotaxis and transport functions resulting from the introduction of an alanine residue at position 134 was suppressed by an additional isoleucine to threonine mutation at residue 132. An x-ray structure of the I132T/G134A double mutant protein (2.2-A resolution) showed that the changes in local structure were accompanied by a diffuse pattern of structural changes in the surrounding region, implying that the suppression derives from a combination of sources.

Amino Acid Sequence↗

PubMed1994

An evaluation of the use of databases in protein structure refinement.

The speed of electron-density fitting during X-ray structure solution and refinement, and the quality of the protein model resulting, can both be enhanced by the use of databases of main- and side-chain conformations. Three structures are compared in this report, one refined at high resolution (1.7 A), and two at lower resolutions using either the database method (2.4 A resolution) or more traditional empirical electron-density fitting (1.9 A resolution). An analysis of peptide orientation was used as an aid in finding unusual portions of main-chain structure. The fit of side chains to known rotamer conformations was used to help determine the accuracy of these atomic positions. In addition, the use of an objective measure of the fit of structures to electron-density maps was evaluated, both alone and in combination with side-chain conformational information.

Journal Article↗

PubMed1994

Identical mutations at corresponding positions in two homologous proteins with nonidentical effects.

The x-ray structure of a mutant (Gly72 to Asp) of the Escherichia coli ribose-binding protein with altered transport function has been solved and refined to 2.2-A resolution with a conventional R-factor (R-factor = [formula: see text]) of 16.0% and good stereochemistry. Comparison with the wild type ribose-binding protein shows that the structure is disturbed little at the actual mutation site, but quite appreciably in a neighboring loop. Changes in the surface of the protein at the site of mutation, however, seem to explain the functional effects. A corresponding mutation of the related glucose/galactose-binding protein has different structural and functional effects due to the different structural context of the mutation site in that protein. These results are consistent with the concept that these proteins have slightly different ways of interacting with the membrane components in transport and chemotaxis.

Amino Acid Sequence↗

PubMed1994

The 1.9 A x-ray structure of a closed unliganded form of the periplasmic glucose/galactose receptor from Salmonella typhimurium.

The three-dimensional structure of a ligand-free closed form of the glucose/galactose binding protein from Salmonella typhimurium has been determined at a resolution of 1.9 A. The crystallographic R-factor for the refined structure is 17.9%. The model contains all the atoms of the 309 residues of the protein sequence, a calcium ion, and 174 water molecules. The root mean square (r.m.s.) deviations for the whole molecule are: 0.010 A for bond lengths and 2.44 degrees for bond angles, indicating a good stereochemistry for the model. This structure shows that the protein is able to close in the absence of ligand, adopting a conformation similar to the liganded form but slightly more open. Water molecules satisfy the hydrogen bonding ability of the hydrophilic side chains of the binding site in a manner which is reminiscent of the sugars' hydrogen-bonding patterns. Since packing forces are weak, the crystallization event is unlikely to trigger a change from an open to a closed conformation. Instead, the latter must be one of the species in equilibrium in solution which is selected by packing in the crystal lattice.

Bacterial Proteins↗

PubMed1994

Planar stacking interactions of arginine and aromatic side-chains in proteins.

A parallel stacking arrangement of the guanidinium groups of arginines directly over the center of the rings of aromatic side-chains is observed much more frequently in proteins than would be expected by chance. This type of interaction, which is often found in locations critical to the function, apparently serves to orient the arginine side-chain without interfering with its ability to form hydrogen bonds elsewhere. It is distinct from the interactions which involve the side-chains of asparagine or glutamine, which do frequently assume a nearly planar relationship to the ring, but at a position at or beyond the ring edge.

Amino Acids↗

PubMed1993

The 1.7 A refined X-ray structure of the periplasmic glucose/galactose receptor from Salmonella typhimurium.

The X-ray structure of the periplasmic glucose/galactose receptor (binding protein) of Salmonella typhimurium (GBP-S) has been refined at 1.7 A resolution with an R-factor of 19.0%. The model contains all 309 residues of the amino acid sequence, 153 water molecules, a calcium ion and beta-D-galactose. The protein consists of two very similar structural domains, each of which is composed a core of parallel beta-sheet flanked on both sides by alpha-helices. Three short stretches of amino acid chain (from symmetrically related portions of the structure) link the domains, and presumably act as a hinge to allow their relative movement in functionally important conformational changes. Galactose is bound between the domains, interacting with a number of side-chains from the loops lining the binding cleft. A combination of hydrogen bonding, hydrophobic and steric effects give rise to tight binding (dissociation constant 0.2 microM) and high specificity. Of nine hydrogen bonding groups, three are aspartate, three asparagine, one histidine (unprotonated), one arginine and one water, contributing 13 hydrogen bonds in total. Additional residues pack against (primarily) non-polar faces of the sugar molecule. The precise arrangement of the hydrogen bonding and hydrophobic residues results in an enclosed binding site with a shape that is a composite of those of the allowed sugar molecules. It is presumed that ligands bind to a more open form of the receptor that then closes by rotation in the hinge. Comparison with the GBP-S structure solved earlier in complex with glucose shows no significant changes, even for the aspartate residue most directly involved with the different sugars. Comparison with the galactose/glucose receptor of Escherichia coli indicates that these two proteins are very similar in overall structure, with the main difference being a 2 to 3 degrees rotation in the hinge. This difference appears to be the result of different crystal packing for the two proteins; it is likely that both conformations are normally found in solution.

Amino Acid Sequence↗

PubMed1993

Structural analysis of the purine repressor, an Escherichia coli DNA-binding protein.

The purine repressor protein, PurR, is a member of the lac repressor, LacI, family of Escherichia coli DNA-binding proteins that bind DNA via a highly conserved N-terminal helix-turn-helix motif. Additionally, the members of this family display strong sequence homologies between their larger C-terminal effector binding/oligomerization domains. Analysis of the PurR primary and secondary structures reveals that its C-terminal corepressor binding domain is highly homologous to another group of E. coli-binding proteins, the periplasmic binding proteins, particularly to the ribose-binding protein (RBP). The high resolution x-ray structure of RBP allows this protein to serve as a template with which to model the predicted secondary structure of the corepressor binding domain of PurR. Similarly, the N-terminal DNA binding domain of PurR can be modeled using the NMR-determined structure of the corresponding region (residues 1-56) from LacI as a template. Combining the two, results in a description of the likely secondary structure topology of PurR and implicates residues important for corepressor binding and dimerization. CD spectroscopic studies on PurR, its corepressor binding domain and RBP result in secondary structure estimates nearly identical with those obtained by sequence analyses, thereby providing further corroborating physical evidence for this topological assignment.

Amino Acid Sequence↗

PubMed1993

Crystallization and preliminary X-ray analysis of the periplasmic dipeptide binding protein from Escherichia coli.

The periplasmic dipeptide-binding protein from Escherichia coli has been purified, freed of bound endogenous ligands, and crystallized. Crystals of the protein in complex with added dipeptides have been subjected to X-ray analysis. The crystals grow as hexagonal bipyramids or eye-shaped disks which have the symmetry of space group P6(1). The unit cell dimensions are a = b = 183 A, c = 212 A, and the diffraction pattern extends to 3.2 A resolution with a conventional X-ray source.

Bacterial Proteins↗

PubMed1993

Inhibition of posterior capsule opacification: the effect of colchicine in a sustained drug delivery system.

We investigated the effect of colchicine in a sustained drug delivery system on posterior capsule opacification (PCO) in rabbit eyes. A polymer matrix wafer, which diffused colchicine at a steady rate, was implanted in the capsular bag of 34 eyes after the lens material was removed by endocapsular phacoemulsification. Three different drug concentrations were used in the rabbit eyes, which were compared with control eyes containing the polymer matrix wafer without colchicine. The mean PCO score was highest in the control group without colchicine and increased steadily over 12 weeks. The rate of PCO formation in all eyes treated with colchicine was significantly lower than in the control group. There was no statistically significant improvement in PCO inhibition with the higher colchicine dosages. Side effects included inflammatory anterior chamber reaction and corneal and retinal complications and were most notable with the highest drug concentration. Slow release of colchicine reduces PCO formation in the rabbit. The optimal biocompatible dosage must be carefully determined and warrants further investigation.

Animals↗

PubMed1992

Ribose and glucose-galactose receptors. Competitors in bacterial chemotaxis.

The periplasmic ribose and glucose-galactose receptors (binding proteins) of Gram-negative bacteria compete for a common inner membrane receptor in bacterial chemotaxis, as well as being the essential primary receptors for their respective membrane transport systems. The high-resolution structures of the periplasmic receptors for ribose (from Escherichia coli) and glucose or galactose (from both Salmonella typhimurium and E. coli) are compared here to outline some features that may be important in their dual functions. The overall structure of each protein consists of two similar domains, both of which are made up of two non-contiguous segments of amino acid chain. Each domain is composed of a core of beta-sheet flanked on both sides with alpha-helices. The two domains are related to each other by an almost perfect intramolecular axis of symmetry. The ribose receptor is smaller as a result of a number of deletions in its sequence relative to the glucose-galactose receptor, mostly occurring in the loop regions; as a result, this protein is also more symmetrical. Many structural features, including some hydrophobic core interactions, a buried aspartate residue and several unusual turns, are conserved between the two proteins. The binding sites for ligand are in similar locations, and built along similar principles, although none of the specific interactions with the sugars is conserved. A comparison shows further that slightly different rotations relate the domains to each other in the three proteins, with the ribose receptor being the most closed, and the Salmonella glucose-galactose receptor the most open. The primary axis of relative rotation is almost perpendicular to that which describes the intramolecular symmetry in each case. These relative rotations of the domains are accompanied by the sliding of some helices as the structures adjust themselves to relieve strain. The hinges which are responsible for most of these relative domain rotations are very similar in the three proteins, consisting of a symmetrical arrangement of beta-strands and alpha-helices and two conserved water molecules that are critical to the hydrogen bonding in the important interdomain region. A region of high sequence and structural similarity between the ribose and glucose-galactose receptors is also located around the intramolecular symmetry axis, on the opposite side of the proteins from the hinge region. This region is that which is altered most by the relative rotations, and is the location of most of the known mutations which affect chemotaxis and transport in the ribose receptor.

Amino Acid Sequence↗

PubMed1992

1.7 A X-ray structure of the periplasmic ribose receptor from Escherichia coli.

The X-ray structure of the periplasmic ribose receptor (binding protein) of Escherichia coli (RBP) was solved at 3 A resolution by the method of multiple isomorphous replacement. Alternating cycles of refitting and refinement have resulted in a model structure with an R-factor of 18.7% for 27,526 reflections from 7.5 to 1.7 A resolution (96% of the data). The model contains 2228 non-hydrogen atoms, including all 271 residues of the amino acid sequence, 220 solvent atoms and beta-D-ribose. The protein consists of two highly similar structural domains, each of which is composed of a core of parallel beta-sheet flanked on both sides by alpha-helices. The two domains are related to each other by an almost perfect 2-fold axis of rotation, with the C termini of the beta-strands of each sheet pointing toward the center of the molecule. Three short stretches of amino acid chain (from symmetrically related portions of the protein) link these two domains, and presumably act as a hinge to allow relative movement of the domains in functionally important conformational changes. Two water molecules are also an intrinsic part of the hinge, allowing crucial flexibility in the structure. The ligand beta-D-ribose (in the pyranose form) is bound between the domains, held by interactions with side-chains of the interior loops. The binding site is precisely tailored, with a combination of hydrogen bonding, hydrophobic and steric effects giving rise to tight binding (0.1 microM for ribose) and high specificity. Four out of seven binding-site residues are charged (2 each of aspartate and arginine) and contribute two hydrogen bonds each. The remaining hydrogen bonds are contributed by asparagine and glutamine residues. Three phenylalanine residues supply the hydrophobic component, packing against both faces of the sugar molecule. The arrangement of these hydrogen bonding and hydrophobic residues results in an enclosed binding site with the exact shape of the allowed sugar molecules; in the process of binding, the ligand loses all of its surface-accessible area. The sites of two mutations that affect the rate of folding of the ribose receptor are shown to be located near small cavities in the wild-type protein. The cavities thus allow the incorporation of the larger residues in the mutant proteins. Since these alterations would seriously affect the ability of the protein to build the first portion of the hydrophobic core in the first domain, it is proposed that this process is the rate-limiting step in folding of the ribose receptor.

Binding Sites↗

PubMed1990

Mutations in the aspartate receptor of Escherichia coli which affect aspartate binding.

The effects of five mutations at arginines 64, 69, and 73 of the Tar protein were analyzed using swarm assays, aspartate binding in purified membranes, and methylation both in vitro and in vivo. The defects in the responses of these mutant receptors to aspartate were shown to be directly attributable to reduced binding of aspartate to the receptor rather than to defects in their signaling characteristics. Mutations at residues 64, 69, and 73 reduced aspartate binding by factors of greater than 10(-4), 10(-3), and 10(-2), respectively. Once aspartate was bound, the mutants exhibited normal signaling properties. No cooperativity was observed in the coupling of aspartate binding to methylation, indicating that the monomers of the receptor dimer act independently. The in vitro methylation system was thus shown to be an effective way of measuring aspartate binding constants and examining the functional integrity of the proteins. The maltose responses of the receptor proteins were affected slightly, or not at all, in an in vivo methylation assay. Two models for the roles of these arginine residues in receptor function are discussed.

Aspartic Acid↗

PubMed1990

Preliminary X-ray data for the periplasmic ribose receptor from Escherichia coli.

Crystals of the periplasmic ribose receptor for chemotaxis and transport in Escherichia coli have been examined by X-ray analysis. The crystals grow as elongated rectangular prisms with the symmetry of the orthorhombic space group P2(1)2(1)2(1). The unit cell dimensions are a = 74.6 A, b = 88.8 A and c = 40.1 A. There is one molecule of molecular weight 28,500 per asymmetric unit.

Carrier Proteins↗

PubMed1987

Additive and independent responses in a single receptor: aspartate and maltose stimuli on the tar protein.

The aspartate and maltose responses of E. coli are mediated through a single membrane receptor, yet the responses are independent and additive. Both stimuli cause methylation of the same 4 glutamic acid residues. More extensive methylation occurs when a cell that has adapted to one stimulus is exposed to the second, or when both stimuli are added simultaneously. The degree of methylation, as well as receptor migration on two-dimensional gels, demonstrates that only one type of protein is involved, rather than two different receptors arising from differential processing of a single gene. A conformational "push-pull" mechanism in which binding of stimulus and covalent modification, producing opposing stresses, can explain these diverse results.

ATP-Binding Cassette Transporters↗