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Journal Issue - Volume 12 Issue 7 (July 2003)

Abstract The fusion of a protein of interest to a large‐affinity tag, such as the maltose‐binding protein (MBP), thioredoxin (TRX), or glutathione‐S‐transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the...

Abstract Two‐dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the β‐helical Tenebrio molitor antifreeze protein (TmAFP) at low temperatures. From measurement of the 3Jαβ1H‐1H scalar coupling constants, the χ1 angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice‐binding face of the protein adopt a preferred rotameric...

Abstract Acid‐sensing ion channels (ASICs) are thought to be important ion channels, particularly for the perception of pain. Some of them may also contribute to synaptic plasticity, learning, and memory. Psalmotoxin 1 (PcTx1), the first potent and specific blocker of the ASIC1a proton‐sensing channel, has been successfully expressed in the Drosophila melanogaster S2 cell recombinant expression system used here for the first time to produce a...

Abstract Phage display enables the presentation of a large number of peptides on the surface of phage particles. Such libraries can be tested for binding to target molecules of interest by means of affinity selection. Here we present SiteLight, a novel computational tool for binding site prediction using phage display libraries. SiteLight is an algorithm that maps the 1D peptide library onto a three‐dimensional (3D) protein surface....

Abstract An approach to discover sequence patterns characteristic of ligand classes is described and applied to aminergic G protein–coupled receptors (GPCRs). Putative ligand‐binding residue positions were inferred from considering three lines of evidence: conservation in the subfamily absent or underrepresented in the superfamily, any available mutation data, and the physicochemical properties of the ligand. For aminergic GPCRs,...

Abstract Numerous X‐ray crystal structures of the metallo‐β‐lactamase from Bacteroides fragilis and related organisms show a β‐hairpin loop immediately adjacent to the active‐site zinc atom(s). Both crystallographic and NMR information show that the end of this β‐hairpin loop, which contains a solvent exposed tryptophan residue, Trp49, is highly flexible in the absence of substrates or other ligands, giving rise in some of the X‐ray structures...

Abstract NMR studies of the binding of a substrate to an inactive HIV‐1 protease construct, containing an active site mutation PRD25N, are reported. Substrate titration measurements monitored by HSQC spectra and a 15N‐edited NOESY experiment show that the chromogenic substrate analog of the capsid/p2 cleavage site binds to PRD25N with an equilibrium dissociation constant, KD, of 0.27 ± 0.05 mM, and upper limits of the association and dissociation rate constants, 2×104...

Abstract Binding of the product inhibitor p‐nitrophenol to the monoclonal esterolytic antibody NPN43C9 has been investigated by performing NMR spectroscopy of the heterodimeric variable‐domain fragment (Fv) of the antibody in the presence and absence of inhibitor. Structural information from changes in chemical shift upon binding has been related to the changes in local dynamics in the active site of the catalytic antibody using NMR...

Abstract We determined the 1.17 Å resolution X‐ray crystal structure of a hybrid peptide based on sequences from coiled‐coil regions of the proteins GCN4 and cortexillin I. The peptide forms a parallel homodimeric coiled‐coil, with Cα backbone geometry similar to GCN4 (rmsd value 0.71 Å). Three stabilizing interactions have been identified: a unique hydrogen bonding–electrostatic network not previously observed in coiled‐coils, and two other...

Abstract We analyzed the total, hydrophobic, and hydrophilic accessible surfaces (ASAs) of residues from a nonredundant bank of 587 3D structure proteins. In an extended fold, residues are classified into three families with respect to their hydrophobicity balance. As expected, residues lose part of their solvent‐accessible surface with folding but the three groups remain. The decrease of accessibility is more pronounced for...

Abstract Sixty‐five families of glycosyltransferases (EC 2.4.x.y) have been recognized on the basis of high‐sequence similarity to a founding member with experimentally demonstrated enzymatic activity. Although distant sequence relationships between some of these families have been reported, the natural history of glycosyltransferases is poorly understood. We used iterative searches of sequence databases, motif extraction,...

Abstract S‐adenosyl‐L‐methionine‐dependent methyltransferases (MTs) are abundant, and highly conserved across phylogeny. These enzymes use the cofactor AdoMet to methylate a wide variety of molecular targets, thereby modulating important cellular and metabolic activities. Thermotoga maritima protein 0872 (TM0872) belongs to a large sequence family of predicted MTs, ranging phylogenetically from relatively simple bacteria to humans....

Abstract Glutamine synthetase (GS) is the key enzyme responsible for the primary assimilation of ammonium in all living organisms, and it catalyses the synthesis of glutamine from glutamic acid, ATP, and ammonium. One of the recently discovered mechanisms of GS regulation involves protein‐protein interactions with a small 65‐residue‐long protein named IF7. Here, we study the structure and stability of IF7 and its binding properties...

Abstract Pyridoxine 5′‐phosphate oxidase catalyzes the terminal step in the synthesis of pyridoxal 5′‐phosphate. The cDNA for the human enzyme has been cloned and expressed in Escherichia coli. The purified human enzyme is a homodimer that exhibits a low catalytic rate constant of ∼0.2 sec−1 and Km values in the low micromolar range for both pyridoxine 5′phosphate and pyridoxamine 5′‐phosphate. Pyridoxal 5′‐phosphate is an effective product...

Abstract We have determined the crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima, TM0651 (gi 4981173), at 2.2 Å resolution by selenomethionine single‐wavelength anomalous diffraction (SAD) techniques. TM0651 is a member of the haloacid dehalogenase (HAD) superfamily, with sequence homology to trehalose‐6‐phosphate phosphatase and sucrose‐6F‐phosphate phosphohydrolase. Selenomethionine labeled...

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