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At least 631 records · Page 35Linked to original sources
PubMed2000

Alpha/Beta-hydrolase fold enzymes: structures, functions and mechanisms.

The alpha/beta-hydrolase fold family of enzymes is rapidly becoming one of the largest group of structurally related enzymes with diverse catalytic functions. Members in this family include acetylcholinesterase, dienelactone hydrolase, lipase, thioesterase, serine carboxypeptidase, proline iminopeptidase, proline oligopeptidase, haloalkane dehalogenase, haloperoxidase, epoxide hydrolase, hydroxynitrile lyase and others. The enzymes all have a Nucleophile-His-Acid catalytic triad evolved to efficiently operate on substrates with different chemical composition or physicochemical properties and in various biological contexts. For example, acetylcholine esterase catalyzes the cleavage of the neurotransmitter acetylcholine, at a rate close to the limits of diffusion of substrate to the active site of the enzyme. Dienelactone hydrolase uses substrate-assisted catalysis to degrade aromatic compounds. Lipases act adsorbed at the water/lipid interface of their neutral water-insoluble ester substrates. Most lipases have their active site buried under secondary structure elements, a flap, which must change conformation to allow substrate to access the active site. Thioesterases are involved in a multitude of biochemical processes including bioluminiscence, fatty acid- and polyketide biosynthesis and metabolism. Serine carboxypeptidases recognize the negatively charged carboxylate terminus of their peptide substrates. Haloalkane dehalogenase is a detoxifying enzyme that converts halogenated aliphatics to the corresponding alcohols, while haloperoxidase catalyzes the halogenation of organic compounds. Hydroxynitrile lyase cleaves carbon-carbon bonds in cyanohydrins with concomitant hydrogen cyanide formation as a defense mechanism in plants. This paper gives an overview of catalytic activities reported for this family of enzymes by discussing selected examples. The current state of knowledge of the molecular basis for catalysis and substrate specificity is outlined. Relationships between active site anatomy, topology and conformational rearrangements in the protein molecule is discussed in the context of enzyme mechanism of action.

Animals↗
PubMed2002

Important amino acid properties for determining the transition state structures of two-state protein mutants.

Understanding the mechanism in the folding pathways of proteins is an important problem in molecular biology. The Phi-value analysis provides insight into the transition state structures during protein folding. In this work, we have analyzed the relationship between the observed Phi values upon mutations in two-state proteins (FK506 binding protein, chymotrypsin inhibitor and src SH3 domain) and the changes in 48 various physico-chemical, energetic and conformational properties. We found that the classification of mutations based on solvent accessibility improved the correlation significantly. The relationship between conformational properties and Phi values determines the presence/absence of secondary structures in the transition state. In buried mutations, the physical properties volume, shape and flexibility, and the thermodynamic properties enthalpy, entropy and free-energy change have significant correlation with Phi. The short and medium-range non-bonded energy in partially buried mutations and average long-range contacts in exposed mutations showed a strong correlation with Phi values. Multiple regression analysis incorporating combinations of three properties from among all possible combinations of the 48 properties increased the correlation coefficient up to 0.99, by an average rise of 20% for all the data sets. Information about local sequence and structure is more important in surface mutations than those in buried mutations for explaining the transition state structures of two-state proteins. Further, the implications of our results for understanding the process of protein folding have been discussed.

Amino Acid Sequence↗
PubMed2004

Channel gating of the glycine receptor changes accessibility to residues implicated in receptor potentiation by alcohols and anesthetics.

The glycine receptor is a target for both alcohols and anesthetics, and certain amino acids in the alpha1 subunit transmembrane segments (TM) are critical for drug effects. Introducing larger amino acids at these positions increases the potency of glycine, suggesting that introducing larger residues, or drug molecules, into the drug-binding cavity facilitates channel opening. A possible mechanism for these actions is that the volume of the cavity expands and contracts during channel opening and closing. To investigate this hypothesis, mutations for amino acids in TM1 (I229C) and TM2 (G256C, T259C, V260C, M263C, T264C, S267C, S270C) and TM3 (A288C) were individually expressed in Xenopus laevis oocytes. The ability of sulfhydryl-specific alkyl methanethiosulfonate (MTS) compounds of different lengths to covalently react with introduced cysteines in both the closed and open states of the receptor was determined. S267C was accessible to short chain (C3-C8) MTS in both open and closed states, but was only accessible to longer chain (C10-C16) MTS compounds in the open state. Reaction with S267C was faster in the open state. I229C and A288C showed state-dependent reaction with MTS only in the presence of agonist. M263C and S270C were also accessible to MTS labeling. Mutated residues more intracellular than M263C did not react, indicating a floor of the cavity. These data demonstrate that the conformational changes accompanying channel gating increase accessibility to amino acids critical for drug action in TM1, TM2, and TM3, which may provide a mechanism by which alcohols and anesthetics can act on glycine (and likely other) receptors.

Alcohols↗
PubMed2005

SuperDrug: a conformational drug database.

MOTIVATION: Different resources exist for experimentally determined and computed three-dimensional (3D)-structures of low molecular weight structures but for approved drugs, no free, publicly accessible source of 3D-structures and conformers is available. Furthermore, for selection purposes or for correlation of structural similarity with medical application, the assignment of the Anatomical Therapeutic Chemical (ATC) classification codes to each structure according to the WHO-scheme would be desirable. RESULTS: The database contains approximately 2500 3D-structures of active ingredients of essential marketed drugs. To account for structural flexibility they are represented by 10(5) structural conformers. Here we present a web-query system enabling searches for drug name, synonyms, trade name, trivial name, formula, CAS-number, ATC-code etc. 2D-similarity screening (Tanimoto coefficients) and an automatic 3D-superposition procedure based on conformational representation are implemented. Drug structures above a similarity threshold as well as superimposed conformers can be retrieved in the mol- file format via a graphical interface. AVAILABILITY: For academic use the system is accessible at http://bioinf.charite.de/superdrug. The retrieval system requires the free browser-plugin 'chime' from MDL for visualization.

Computer Graphics↗
PubMed1977

Molecular internalization of a region of myelin basic protein.

The conformation of myelin encephalitogenic or basic protein (BP) was investigated with a double-antibody radioimmunoassay by studying the reaction of BP or its fragments with antibodies produced in two rabbits against peptide 43-88 linked to rabbit serum albumin. Both antisera reacted well with peptide 43-88 but showed little or no reaction with BP. Absorption of these antisera with a BP-immunoadsorbent did not remove the antibody activity against peptide 43-88. Within the region of peptide 43- 88 it was shown that peptides 68-88 and 79-88 gave an equivalent or better reaction than peptide 43-88, whereas peptides 43-67 and 64-73 had very little reactivity. In the BP fragments containing region 43-88, peptide 1-88 showed the best reactivity, peptide 20-166 showed minimal reactivity, while peptide 1-115 showed none. These data document the internal position of at least a portion of peptide 43-88 and all of residues 79-88 in the BP molecule. The much greater reactivity of peptide 1-88 as compared to peptide 1-115 suggests that the region or a portion of the region of BP containing residues 89- 115 participates in the conformational alignment of BP restricting access to peptide 79-88. After absorption with BP, neither of the antisera prepared to peptide 43-88 reacted with PNS myelin in fixed tissue sections but continued to react with CNS myelin in similarly treated sections. The present findings demonstrate the need to consider the role of shielded antigenic determinants in the investigation of antigens or of immune responses.

Animals↗
PubMed2002

Head-to-backbone cyclization of peptides on solid support by nucleophilic aromatic substitution.

A new versatile synthetic route is presented for the cyclization of tripeptides on solid support using nucleophilic aromatic substitution in the cyclization step. Identification of all conformers within a limit of 3 kcal/mol from the identified global minimum conformations by Monte Carlo conformational searching reveals that five out of six synthesized compounds have well-defined peptide backbone conformational properties. This was determined by clustering the identified conformers against a filter of seven to nine torsion angles in the peptide backbone. Thus, the results meet our goal to find synthetic routes to peptides that are conformationally sufficiently locked to serve as convenient leads for further development of pharmacophoric models. The strategy is based on Fmoc-peptide chemistry on a N-aminoethyl-substituted glycine bound to the commercially available Rink amide PS-resin. After deprotection of the N-terminus of the tripeptide, it is acylated with a fluoronitrobenzoic acid. Subsequently, a Boc group on the N-bound aminoethyl substituent is selectively deprotected allowing cyclization from the head (N-terminus) to the backbone substituent, thereby leading to the desired cyclized tripeptides. A number of representative examples of peptides cyclized by this method have been synthesized and characterized by NMR. Protecting groups that allow the incorporation of side chain functionalized amino acids have been found. Thus, the route provides access to generic libraries of conformationally restricted peptide sequences expressing a range of proteinogenic pharmacophores.

Catalysis↗
PubMed2002

Approaches toward the directed design of a vaccine against Borrelia burgdorferi.

The overall efficacy of a recombinant vaccine for Lyme disease that is effective worldwide will depend upon the selection of one or more immunoprotective target(s) and the frequency of genetic variation, which can alter the antigenicity of the immunoprotective epitopes of the target proteins. Careful delineation of these protective epitopes on target antigens is essential for the development of vaccine candidates as well as for understanding the limitations of such vaccines. Structural models of these targets will provide critical information about conformation and specific residue surface accessibility for defining protective epitopes. Co-crystal structures with Fab fragments of protective antibodies will further delineate critical antigen surfaces. Population genetics will provide vital information on the heterogeneity of these proteins. Detailed epitope mapping will provide the information needed for the bioengineering of antigens needed to expand the specificity of a candidate vaccine.

Antigens, Surface↗
PubMed2005

A structural model for the osmosensor, transporter, and osmoregulator ProP of Escherichia coli.

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily (MFS), acts as an osmosensor and an osmoregulator in cells and after purification and reconstitution in proteoliposomes. H(+)-osmoprotectant symport via ProP is activated when medium osmolality is elevated with membrane impermeant osmolytes. The three-dimensional structure of ProP was modeled with the crystal structure of MFS member GlpT as a template. This GlpT structure represents the inward (or cytoplasm)-facing conformation predicted by the alternating access model for transport. LacZ-PhoA fusion analysis and site-directed fluorescence labeling substantiated the membrane topology and orientation predicted by this model and most hydropathy analyses. The model predicts the presence of a proton pathway within the N-terminal six-helix bundle of ProP (as opposed to the corresponding pathway found within the C-terminal helix bundle of its paralogue, LacY). Replacement of residues within the N-terminal helix bundle impaired the osmotic activation of ProP, providing the first indication that residues outside the C-terminal domain are involved in osmosensing. Some residues that were accessible from the periplasmic side, as predicted by the structural model, were more susceptible to covalent labeling in permeabilized membrane fractions than in intact bacteria. These residues may be accessible from the cytoplasmic side in structures not represented by our current model, or their limited exposure in vivo may reflect constraints on transporter structure that are related to its osmosensory mechanism.

Alkaline Phosphatase↗
PubMed2007

Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors.

The multidrug exporter AcrB is the inner membrane component of the AcrAB-TolC drug efflux system in Escherichia coli and is responsible for the resistance of this organism to a wide range of drugs. Here we describe the crystal structure of the trimeric AcrB in complex with a designed ankyrin-repeat protein (DARPin) inhibitor at 2.5-A resolution. The three subunits of AcrB are locked in different conformations revealing distinct channels in each subunit. There seems to be remote conformational coupling between the channel access, exit, and the putative proton-translocation site, explaining how the proton motive force is used for drug export. Thus our structure suggests a transport pathway not through the central pore but through the identified channels in the individual subunits, which greatly advances our understanding of the multidrug export mechanism.

Ankyrin Repeat↗
PubMed1983

Conformational characteristics of the dinucleoside triphosphate pCpGp from energy-minimization studies.

The influence of the 3'- and 5'- terminal phosphates on the conformational characteristics of the dinucleoside monophosphate CpG is described in this paper. The computed potential energy of the system is minimized with respect to the relevant 10 dihedral angles permitting the two sugar rings to adopt the alternative puckering states, 2E and 3E. Of the 84 conformations considered, 22 become energetically accessible. The familiar A-, B-, Z- and Watson-Crick-type backbone states of DNA subunits become low-energy forms for this RNA unit pCpGp also. The Watson-Crick-type backbone is invariably preferred in all the four sugar pucker sequences, indicating its importance in the dynamics of sugar pucker fluctuations and in the DNA-RNA association. The interphosphate geometries and the possible hydrogen-bonded states are discussed in relation to the varied folded/extended polynucleotide structures.

Calorimetry↗
PubMed1997

Construction and characterization of a radio-iodinatable mutant of recombinant human CD4.

Recombinant soluble human CD4 (rsCD4) has been used in iodinated form to study the interaction of CD4 with its ligands. However, the utility of [125I]-rsCD4 is limited because rsCD4 is inefficiently iodinated and the iodinated protein is poorly active. The iodination properties of rsCD4 most likely reflect the poor accessibility of the tyrosine residues, apparent from the available X-ray structures. We have generated an iodinatable mutant of rsCD4 by substituting Tyr for Phe(179) in the flexible, solvent-exposed C-terminal region of rsCD4(183), a truncated form of CD4 that consists of the first 183 residues of CD4 and includes the binding sites for HIV-1 gp120 and MHC class II molecules. When F179Y rsCD4(183) is iodinated under trace-labeling conditions, the efficiency of 125I incorporation and the percentage of iodinated molecules that are active are much enhanced compared with WT rsCD4. Moreover, trace-labeled [125I]-F179Y rsCD4(183) has the same affinity for HIV-1 rgp120 as unlabeled WT rsCD4. The improved activity of trace-labeled [125I]-F179Y rsCD4(183) appears to be due to effective competition by Y179 for reactive iodine species that, in WT rsCD4, react with traces of denatured protein and/or with residues critical for activity or conformational integrity. The incorporation of accessible tyrosine residues may improve the iodinatibility of a protein both by introducing a readily iodinatable residue and by protecting sensitive proteins from adverse reactions.

Animals↗
PubMed1975

Enzymic and immunochemical properties of lysozyme. X. Conformation, enzymic activity and immunochemistry of lysozyme reduced at two carboxyl groups.

Reduction of lysozyme by diborane, followed by air oxidation of the reduced disulfides and chromatography on CM-cellulose, yielded a homogeneous derivative. In the derivative, the carboxyl groups of aspartic acid 119 and the end-chain leucine residue were reduced to their corresponding alcohols. Correct re-forming of the disulfide bonds was demonstrated by peptide mapping of the tryptic hydrolysates of the derivative and lysozyme without breaking the disulfide bonds, followed by identification of the disulfide-containing peptides. Correct disulfide pairing in the two-disulfide peptide in the tryptic hydrolysate was established from its immunochemical behavior. Preparations of the two-disulfide fragment from lysozyme and derivative had equal inhibitory activities (26 or 32%) of the reaction of lysozyme with two homologous antisera. In ORD measurements, lysozyme and the derivative had equal rotatory powers at neutral pH. However, the bo value for the derivative decreased by about 10%. Below pH 6.4 and above pH 8.0, the derivative was less rotatory than native lysozyme. In CD measurements at neutral pH, the negative ellipticity bands at 220 and 208 nm showed little or no decrease in the derivative relative to the native protein. Although conformational differences between the derivative and its parent protein were almost undetectable by ORD and CD measurements, they were readily detected by chemical monitoring of the conformation. In the derivative, both accessibility to tryptic hydrolysis and reducibility of the disulfide bonds increased markedly. The enzymic activity of the derivative was decreased but retained the same pH optimum. With antisera to lysozyme or antisera to the derivative, lysozyme and its derivative possessed equal antigenic reactivities. The immunochemical findings further confirm the correct refolding of the disulfides. Also, they indicate that aspartic acid 119 and the C-terminal leucine residue are not part of an antigenic reactive region in lysozyme.

Amino Acids↗
PubMed2004

SCit: web tools for protein side chain conformation analysis.

SCit is a web server providing services for protein side chain conformation analysis and side chain positioning. Specific services use the dependence of the side chain conformations on the local backbone conformation, which is described using a structural alphabet that describes the conformation of fragments of four-residue length in a limited library of structural prototypes. Based on this concept, SCit uses sets of rotameric conformations dependent on the local backbone conformation of each protein for side chain positioning and the identification of side chains with unlikely conformations. The SCit web server is accessible at http://bioserv.rpbs.jussieu.fr/SCit.

Amino Acids↗
PubMed1999

NMR solution structure of alpha-conotoxin ImI and comparison to other conotoxins specific for neuronal nicotinic acetylcholine receptors.

Alpha-Conotoxins, peptides produced by predatory species of Conus marine snails, are potent antagonists of nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels involved in synaptic transmission. We determined the NMR solution structure of the smallest known alpha-conotoxin, ImI, a 12 amino acid peptide that binds specifically to neuronal alpha7-containing nAChRs in mammals. Calculation of the structure was based on a total of 80 upper distance constraints and 31 dihedral angle constraints resulting in 20 representative conformers with an average pairwise rmsd of 0.44 A from the mean structure for the backbone atoms N, Calpha, and C' of residues 2-11. The structure of ImI is characterized by two compact loops, defined by two disulfide bridges, which form distinct subdomains separated by a deep cleft. Two short 310-helical regions in the first loop are followed by a C-terminal beta-turn in the second. The two disulfide bridges and Ala 9 form a rigid hydrophobic core, orienting the other amino acid side chains toward the surface. Comparison of the three-dimensional structure of ImI to those of the larger, 16 amino acid alpha-conotoxins PnIA, PnIB, MII, and EpI-also specific for neuronal nAChRs-reveals remarkable similarity in local backbone conformations and relative solvent-accessible surface areas. The core scaffold is conserved in all five conotoxins, whereas the residues in solvent-exposed positions are highly variable. The second helical region, and the specific amino acids that the helix exposes to solvent, may be particularly important for binding and selectivity. This comparative analysis provides a three-dimensional structural basis for interpretation of mutagenesis data and structure-activity relationships for ImI as well other neuronal alpha-conotoxins.

Amino Acid Sequence↗
PubMed2006

Model for proton transport coupled to protein conformational change: application to proton pumping in the bacteriorhodopsin photocycle.

A modeling method is presented for protein systems in which proton transport is coupled to conformational change, as in proton pumps and in motors driven by the proton-motive force. Previously developed methods for calculating pKa values in proteins using a macroscopic dielectric model are extended beyond the equilibrium case to a master-equation model for the time evolution of the system through states defined by ionization microstate and a discrete set of conformers. The macroscopic dielectric model supplies free energy changes for changes of protonation microstate, while the method for obtaining the energetics of conformational change and the relaxation rates, the other ingredients needed for the master equation, are system dependent. The method is applied to the photoactivated proton pump, bacteriorhodopsin, using conformational free energy differences from experiment and treating relaxation rates through three adjustable parameters. The model is found to pump protons with an efficiency relatively insensitive to parameter choice over a wide range of parameter values, and most of the main features of the known photocycle from very early M to the return to the resting state are reproduced. The boundaries of these parameter ranges are such that short-range proton transfers are faster than longer-range ones, which in turn are faster than conformational changes. No relaxation rates depend on conformation. The results suggest that an "accessibility switch", while not ruled out, is not required and that vectorial proton transport can be achieved through the coupling of the energetics of ionization and conformational states.

Bacteriorhodopsins↗
PubMed2000

Highly stable oligomerization forms of HIV-1 Tat detected by monoclonal antibodies and requirement of monomeric forms for the transactivating function on the HIV-1 LTR.

The use of newly generated murine monoclonal antibodies directed against distinct epitopes of a functionally active, chemically synthesized HIV-1 Tat protein has permitted the identification of several molecular forms including monomers, dimers and trimers. Dimers and trimers are particularly stable and resistant to strong reducing conditions. Through epitope mapping it has been possible to demonstrate that the major immunodominant epitope is contained within the basic region of the Tat protein and is lost after oligomerization of the molecule. In contrast, N-terminal, C-terminal and conformation-dependent epitopes are still accessible to mAb specific recognition after Tat oligomerization. Moreover, by using a quantitative HIV-LTR transactivation assay depending upon exogenous Tat, we could extrapolate the amount of functional Tat produced by cell lines stably transfected with the viral transactivator. More importantly, we could show that only the monomeric form of exogenous Tat is the relevant functional form acting in cells harbouring the HIV-1 LTR promoter.

Alkylation↗
PubMed2000

Crystal structure of pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases.

The x-ray structure of the lipase from Pseudomonas aeruginosa PAO1 has been determined at 2.54 A resolution. It is the first structure of a member of homology family I.1 of bacterial lipases. The structure shows a variant of the alpha/beta hydrolase fold, with Ser(82), Asp(229), and His(251) as the catalytic triad residues. Compared with the "canonical" alpha/beta hydrolase fold, the first two beta-strands and one alpha-helix (alphaE) are not present. The absence of helix alphaE allows the formation of a stabilizing intramolecular disulfide bridge. The loop containing His(251) is stabilized by an octahedrally coordinated calcium ion. On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region. A triacylglycerol analogue is covalently bound to Ser(82) in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains. The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction. Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme.

Amino Acid Sequence↗
PubMed2002

Calcium-dependent homoassociation of E-cadherin by NMR spectroscopy: changes in mobility, conformation and mapping of contact regions.

Cadherins are calcium-dependent cell surface proteins that mediate homophilic cellular adhesion. The calcium-induced oligomerization of the N-terminal two domains of epithelial cadherin (ECAD12) was followed by NMR spectroscopy in solution over a large range of protein (10 microM-5 mM) and calcium (0-5 mM) concentrations. Several spectrally distinct states could be distinguished that correspond to a calcium-free monomeric form, a calcium-bound monomeric form, and to calcium-bound higher oligomeric forms. Chemical shift changes between these different states define calcium-binding residues as well as oligomerization contacts. Information about the relative orientation and mobility of the ECAD12 domains in the various states was obtained from weak alignment and 15N relaxation experiments. The data indicate that the calcium-free ECAD12 monomer adopts a flexible, kinked conformation that occludes the dimer interface observed in the ECAD12 crystal structure. In contrast, the calcium-bound monomer is already in a straight, non-flexible conformation where this interface is accessible. This mechanism provides a rational for the calcium-induced adhesiveness. Oligomerization induces chemical shift changes in an area of domain CAD1 that is centered at residue Trp-2. These shift changes extend to almost the entire surface of domain CAD1 at high (5 mM) protein concentrations. Smaller additional clusters of shift perturbations are observed around residue A80 in CAD1 and K160 in CAD2. According to weak alignment and relaxation data, the symmetry of a predominantly dimeric solution aggregate at 0.6 mM ECAD12 differs from the approximate C2-symmetry of the crystalline dimer.

Amino Acid Sequence↗