Unlike structures determined by X-ray crystallography, which are deposited in the Brookhaven Protein Data Bank (Abola et al., 1987) as a single structure, each NMR-derived structure is often an ensemble containing many structures, consistent with restraint set used. However, there need to select 'representative' or subset of from such ensemble. This useful, for example, case homology modelling when compiling relational database protein structures. It has been shown that cluster analysis,...

An approach is described for modelling the three-dimensional structure of a protein from tertiary structures several homologous proteins that have been determined by X-ray analysis. A method developed simultaneous superposition molecules and calculation an ‘average structure’ or ‘framework’. Investigation convergence properties this method, in case both weighted unweighted least squares, demonstrates give unique answer latter robust family proteins. Multi-dimensional scaling used to subgroup...

We present an atomic-level description of the reaction chemistry enzyme-catalyzed dominated by proton tunneling. By solving structures intermediates at near-atomic resolution, we have identified pathway for tryptamine oxidation aromatic amine dehydrogenase. Combining experiment and computer simulation, show transfer occurs predominantly to oxygen O2 Asp(128)beta in a tunneling over approximately 0.6 angstroms. The role long-range coupled motions promoting is controversial. that, this enzyme...

Enzymatic breakage of the substrate C−H bond by Methylophilus methyltrophus (sp. W3A1) methylamine dehydrogenase (MADH) has been studied stopped-flow spectroscopy. The rate reduction tryptophan tryptophylquinone (TTQ) cofactor a large kinetic isotope effect (KIE = 16.8 ± 0.5), and KIE is independent temperature. Analysis temperature dependence revealed that extreme (ground state) quantum tunneling responsible for transfer hydrogen nucleus. Reaction rates are strongly dependent on...

This paper describes a rapid, automated procedure which can be used for model building sidechains using (i) spatial information from in topologically equivalent positions as far such correlation is observed, and then (ii) most probable conformations of the respective secondary structure type. Analysis residues structurally conserved regions family proteins implies that atoms rather than should considered when building. Rules modelling all 20 each other α-helical, β-Sheet loop regions—a total...

1. We describe the effects on channel function of changing an aspartate residue (Asp172) in a membrane‐spanning alpha‐helix murine inward rectifier, IRK1, by site‐directed mutagenesis. 2. Alteration Asp172 to Glu (charged) or Gln Asn (polar but uncharged) produced functional channels showing rectification, though rectification was weaker with and Asn. 3. Intrinsic gating around potassium equilibrium potential, EK, conserved only if charge 172 conserved. Currents through this position showed...

Substitution of beta3 residue Asp119, Ser121, or Ser123 results in a loss the ligand binding function integrin alphaIIbbeta3. Homologous residues other beta subunits are similarly critical for function. This DXSXS motif is also present I domain certain alpha subunits, where it constitutes portion unique metal ion-dependent adhesion site (MIDAS). In this report, we have utilized crystal structure recombinant alphaM to produce three-dimensional model homologous region subunit. We performed...

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its agonistic antibodies, which are currently in early clinical trials for treating various malignancies, induce apoptosis through triggering of either TRAIL-R1 or TRAIL-R2. Based on studies using monoclonal we recently proposed that primary chronic lymphocytic leukemic cells seem to signal primarily TRAIL-R1. We have now synthesized mutant forms TRAIL specific The selectivity these mutants cell lines is due selective...

The cytochrome P450 responsible for the debrisoquine/sparteine polymorphism (P450 2D6) has been produced in large quantities by expression of a modified cDNA baculovirus. A polyhistidine extension was incorporated at C-terminus expressed protein, which, after purification protein on nickel-agarose column, could be removed proteolytically treatment with thrombin. Purified yields 2D6 were 2.4 mg from 700 mL cell culture. had greater than 90% heme content and fully active, having no residual...

Phenylalanine 87 of Bacillus megaterium cytochrome P450 BM3, a residue close to the heme in substrate binding pocket, has been replaced by alanine site-directed mutagenesis. The substitution had no effect on rate hydroxylation laurate and increased affinity for both intact enzyme its domain 2.6-6-fold ferric state. NMR paramagnetic relaxation measurements showed that initial enzyme-substrate complex, where binds relatively far from heme, position or orientation bound substrate. However, next...

Three-bond 3JHN alpha coupling constants have been determined for 106 residues and 3J beta measured 57 of the 129-residue protein hen egg white lysozyme. These NMR data compared with torsion angles defined in tetragonal triclinic crystal forms protein. For most values were consistent phi found both forms; RMS difference between calculated by using structure experimental is 0.88 Hz. Thus there appears to be no significant averaging angle either interior or at surface 41 where determined, are...

Abstract Three CYP6Z genes are linked to a major pyrethroid resistance locus in the mosquito Anopheles gambiae . We have expressed CYP6Z2 Escherichia coli and produced structural model order examine its role detoxification. E. membranes co‐expressing An. P450 reductase (AgCPR) catalysed dealkylation of benzyloxyresorufin with kinetic parameters K m = 0.13 µM; cat 1.5 min ‐1 The IC 50 values wide range compounds were measured. Pyrethroids cypermethrin permethrin low values, but not...

Abstract Many commonly used, structurally diverse, drugs block the human ether‐a‐go‐go‐related gene (hERG) K + channel to cause acquired long QT syndrome, which can lead sudden death via lethal cardiac arrhythmias. This undesirable side effect is a major hurdle in development of safe drugs. To gain insight about structure hERG and nature drug we have produced structural models pore domain, into each docked set 20 blockers. In absence an experimentally determined three‐dimensional hERG, was...

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to predominantly structural role for loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., Sutcliffe, M. J. (2002) <i>Proteins</i> 49, 216–231) suggest second acidic residue,...

C–H bond breakage by tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) occurs vibrationally assisted tunneling (Basran, J., Sutcliffe, M. and Scrutton, N. S. (1999)Biochemistry 38, 3218–3222). We show here a similar mechanism in TTQ-dependent aromatic amine (AADH). The rate of TTQ reduction dopamine AADH has large, temperature independent kinetic isotope effect (KIE = 12.9 ± 0.2), which is highly suggestive tunneling. H-transfer compromised with benzylamine as...

Structural models have been produced for three types of non-NMDA inotropic glutamate receptors: an AMPA receptor, GluR1, a kainate GluR6; and low-molecular-weight receptor from goldfish, GFKAR alpha. Modeling was restricted to the domains proteins that bind neurotransmitter form ion channel. Model building combined homology modeling, distance geometry, molecular mechanics, interactive known constraints. The indicate new potential interactions in extracellular domain between protein agonists,...