bluuebunny/crossref_metadata_2025_split huggingface dataset:
Octameric enolase from the hyperthermophilic bacterium Thermotoga maritima: Purification, characterization, and image processing
Authors: Schurig Hartmut , Rutkat Kerstin , Jaenicke Rainer , Rachel Reinhard 
Published: June 2010

AbstractEnolase (2‐phospho‐D‐glycerate hydrolase; EC 4.2.1.11) from the hyperthermophilic bacterium Thermotoga maritima was purified to homogeneity. The N‐terminal 25 amino acids of the enzyme reveal a high degree of similarity to enolases from other sources. As shown by sedimentation analysis and gel‐permeation chromatography, the enzyme is a 345‐kDa homooctamer with a subunit molecular mass of 48 ± 5 kDa. Electron microscopy and image processing yield ring‐shaped particles with a diameter of 17 nm and fourfold symmetry. Averaging of the aligned particles proves the enzyme to be a tetramer of dimers. The enzyme requires divalent cations in the activity assay, Mg2+ being most effective. The optimum temperature for catalysis is 90 °C, the temperature dependence yields a nonlinear Arrhenius profile with limiting activation energies of 75 kJ mol−1 and 43 kJ mol−1 at temperatures below and above 45 °C. The pH optimum of the enzyme lies between 7 and 8. The apparent Km values for 2‐phospho‐D‐glycerate and Mg2+ at 75 °C are 0.07 mM and 0.03 mM; with increasing temperature, they are decreased by factors 2 and 30, respectively. Fluoride and phosphate cause competitive inhibition with a Ki of 0.14 mM. The enzyme shows high intrinsic thermal stability, with a thermal transition at 90 and 94 °C in the absence and in the presence of Mg2+.
Weakening of the interface between adjacent catalytic chains promotes domain closure in escherichia coli aspartate transcarbamoylase
Authors: Baker Darren P. , Keiser Richard T. , Kantrowitz Evan R. , Fetler Luc , Vachette Patrice 
Published: June 2010

AbstractAspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. Asp‐100, from one catalytic chain, is involved in stabilizing the C1‐C2 interface by means of its interaction with Arg‐65 from an adjacent catalytic chain. Replacement of Asp‐100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 52:10150–10158). In order to determine whether these properties were due to promotion of domain closure induced by the weakening of the C1‐C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp‐100 → Ala mutation was introduced into the Glu‐50 → Ala holoenzyme, a mutant in which domain closure is impaired. The Glu‐50/Asp‐100 → Ala enzyme is fourfold more active than the Glu‐50 → Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate. In addition, the Asp‐100 → Ala mutation restores the ability of the Glu‐50 → Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N‐(phosphonacetyl)‐L‐aspartate (PALA). At subsaturating concentrations of aspartate, the Glu‐50/Asp‐100 → Ala enzyme is activated more by ATP than the Glu‐50 → Ala enzyme and is also inhibited more by CTP than either the wild‐type or the Glu‐50 → Ala enzyme. As opposed to the wild‐type enzyme, the Glu‐50/Asp‐100 → Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Glu‐50/Asp‐100 → Ala enzyme by solution X‐ray scattering indicates that the double mutant exists in the same T quaternary structure as the wild‐type enzyme in the absence of ligands and in the same R quaternary structure in the presence of saturating PALA. However, saturating concentrations of carbamoyl phosphate and succinate only convert a fraction of the Glu‐50/Asp‐100 → Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu‐50 → Ala and wild‐type enzymes. Solution X‐ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu‐50/Asp‐100 → Ala enzyme.
Divergent evolution of a β/α‐barrel subclass: Detection of numerous phosphate‐binding sites by motif search
Authors: Bork Peer , Gellerich Johannes , Groth Holger , Hooft Rob , Martin Falk 
Published: June 2010

AbstractStudy of the most conserved region in many β/α‐barrels, the phosphate‐binding site, revealed a sequence motif in a few β/α‐barrels with known tertiary structure, namely glycolate oxidase (GOX), cytochrome b2 (Cyb2), tryptophan synthase α subunit (TrpA), and the indoleglycerolphosphate synthase (TrpC). Database searches identified this motif in numerous other enzyme families: (1) IMP dehydrogenase (IMPDH) and GMP reductase (GuaC); (2) phosphoribosylformimino‐5‐aminoimidazol carboxamide ribotide isomerase (HisA) and the cyclase‐producing D‐erythro‐imidazole‐glycerolphosphate (HisF) of the histidine biosynthetic pathway; (3) dihydroorotate dehydrogenase (PyrD); (4) glutamate synthase (GltB); (5) ThiE and ThiG involved in the biosynthesis of thiamine as well as related proteins; (6) an uncharacterized open reading frame from Erwinia herbicola; and (7) a glycerol uptake operon antiterminator regulatory protein (GlpP). Secondary structure predictions of the different families mentioned above revealed an alternating order of β‐strands and α‐helices in agreement with a β/α‐barrel‐like topology. The putative phosphate‐binding site is always found near the C‐terminus of the enzymes, which are all at least about 200 amino acids long. This is compatible with its assumed location between strand 7 and helix 8. The identification of a significant motif in functionally diverse enzymes suggests a divergent evolution of at least a considerable fraction of β/α‐barrels. In addition to the known accumulation of β/α‐barrels in the tryptophan biosynthetic pathway, we observe clusters of these enzymes in histidine biosynthesis, purine metabolism, and apparently also in thiamine biosynthesis. The substrates are mostly heterocyclic compounds. Although the marginal sequence similarities do not allow a reconstruction of the barrel spreading, they support the idea of pathway evolution by gene duplication.
Structural basis of substrate specificity in the serine proteases
Authors: Perona John J. , Craik Charles S. 
Published: June 2010

AbstractStructure‐based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system.
Approaches to labeling and identification of active site residues in glycosidases
Authors: Withers Stephen G. , Aebersold Ruedi 
Published: June 2010

AbstractGlycosidases play a key role in a number of biological processes and, as such, are of considerable clinical and bio‐technological importance. Knowledge of the identities of catalytically important active site residues is essential for understanding the catalytic mechanism, for enzyme classification, and for targeted bioengineering of glycosidases with altered characteristics. Here we review and discuss traditional strategies and novel approaches based on tandem mass spectrometry for the identification of the key active site residues in glycosidases.
Conservative and nonconservative mutations in proteins: Anomalous mutations in a transport receptor analyzed by free energy and quantum chemical calculations
Authors: Cannon William R. , Briggs James M. , Shen Jian , McCammon J. Andrew , Quiocho Florante A. 
Published: June 2010

AbstractExperimental studies on a bacterial sulfate receptor have indicated anomalous relative binding affinities for the mutations Ser130 → Cys, Ser130 → Gly, and Ser130 → Ala. The loss of affinity for sulfate in the former mutation was previously attributed to a greater steric effect on the part of the Cys side chain relative to the Ser side chain, whereas the relatively small loss of binding affinity for the latter two mutations was attributed to the loss of a single hydrogen bond. In this report we present quantum chemical and statistical thermodynamic studies of these mutations. Qualitative results from these studies indicate that for the Ser130 → Cys mutation the large decrease in binding affinity is in part caused by steric effects, but also significantly by the differential work required to polarize the Cys thiol group relative to the Ser hydroxyl group. The Gly mutant cobinds a water molecule in the same location as the Ser side chain resulting in a relatively small decrease in binding affinity. Results for the Ala mutant are in disagreement with experimental results but are likely to be limited by insufficient sampling of configuration space due to physical constraints applied during the simulation.
Stability of ribonuclease T2 from Aspergillus oryzae
Authors: Kawata Yasushi , Hamaguchi Kozo 
Published: June 2010

AbstractThe stability of ribonuclease T2 (RNase T2) from Aspergillus oryzae against guanidine hydrochloride and heat was studied by using CD and fluorescence. RNase T2 unfolded and refolded reversibly concomitant with activity, but the unfolding and refolding rates were very slow (order of hours). The free energy change for unfolding of RNase T2 in water was estimated to be 5.3 kcal‐mol−1 at 25 °C by linear extrapolation method. From the thermal unfolding experiment in 20 mM sodium phosphate buffer at pH 7.5, the Tm and the enthalpy change of RNase T2 were found to be 55.3 °C and 119.1 kcal‐mol−1, respectively. From these equilibrium and kinetic studies, it was found that the stability of RNase T2 in the native state is predominantly due to the slow rate of unfolding.
Comparative modeling of the three‐dimensional structure of Type II antifreeze protein
Authors: Sönnichsen Frank D. , Sykes Brian D. , Davies Peter L. 
Published: June 2010

AbstractType II antifreeze proteins (AFP), which inhibit the growth of seed ice crystals in the blood of certain fishes (sea raven, herring, and smelt), are the largest known fish AFPs and the only class for which detailed structural information is not yet available. However, a sequence homology has been recognized between these proteins and the carbohydrate recognition domain of C‐type lectins. The structure of this domain from rat mannose‐binding protein (MBP‐A) has been solved by X‐ray crystallography (Weis WI, Drickamer K, Hendrickson WA, 1992, Nature 560:127–134) and provided the coordinates for constructing the three‐dimensional model of the 129‐amino acid Type II AFP from sea raven, to which it shows 19% sequence identity. Multiple sequence alignments between Type II AFPs, pancreatic stone protein, MBP‐A, and as many as 50 carbohydrate‐recognition domain sequences from various lectins were performed to determine reliably aligned sequence regions. Successive molecular dynamics and energy minimization calculations were used to relax bond lengths and angles and to identify flexible regions. The derived structure contains two α‐helices, two β‐sheets, and a high proportion of amino acids in loops and turns. The model is in good agreement with preliminary NMR spectroscopic analyses. It explains the observed differences in calcium binding between sea raven Type II AFP and MBP‐A. Furthermore, the model proposes the formation of five disulfide bridges between Cys 7 and Cys 18, Cys 35 and Cys 125, Cys 69 and Cys 100, Cys 89 and Cys 111, and Cys 101 and Cys 117. Based on the predicted features of this model, a site for proteinice interaction is proposed.
Stabilization of a strained protein loop conformation through protein engineering
Authors: Hodel Alec , Fox Robert O. , Kautz Roger A. 
Published: June 2010

AbstractStaphylococcal nuclease is found in two folded conformations that differ in the isomerization of the Lys 116‐ Pro 117 peptide bond, resulting in two different conformations of the residue 112–117 loop. Thecisform is favored over thetranswith an occupancy of 90%. Previous mutagenesis studies have shown that when Lys 116 is replaced by glycine, atransconformation is stabilized relative to thecisconformation by the release of steric strain in thetransform. However, when Lys 116 is replaced with alanine, the resulting variant protein is identical to the wild‐type protein in its structure and in the dominance of thecisconfiguration. The results of these studies suggested that any nuclease variant with a non‐glycine residue at position 116 should also favor thecisform because of steric requirements of the β‐carbon at this position. In this report, we present a structural analysis of four nuclease variants with substitutions at position 116. Two variants, K116E and K116M, follow the “β‐carbon” hypothesis by favoring thecisform. Furthermore, the crystal structure of K116E is nearly identical to that of the wild‐type protein. Two additional variants, K116D and K16N, provide exceptions to this simple “β‐carbon” rule in that thetransconformation is stabilized relative to thecisconfiguration by these substitutions. Crystallographic data indicate that this stabilization is effected through the addition of tertiary interactions between the side chain of position 116 with the surrounding protein and water structure. The detailedtransconformation of the K116D variant appears to be similar to thetransconformation observed in the K116G variant, suggesting that these two mutations stabilize the same conformation but through different mechanisms.
Simultaneous modeling of multiple loops in proteins
Authors: Rosenbach Dror , Rosenfeld Rakefet 
Published: June 2010

AbstractThe most reliable methods for predicting protein structure are by way of homologous extension, using structural information from a closely related protein, or by “threading” through a set of predefined protein folds (“inverse folding”). Both sets of methods provide a model for the core of the protein —the structurally conserved secondary structures. Due to the large variability both in sequence and size of the loops that connect these secondary structures, they generally cannot be modeled using these techniques. Loop‐closure algorithms are aimed at predicting loop structures, given their end‐to‐end distance. Various such algorithms have been described, and all have been tested by predicting the structure of a single loop in a known protein. In this paper we propose a method, which is based on the bond‐scaling‐relaxation loop‐closure algorithm, for simultaneously predicting the structures of multiple loops, and demonstrate that, for two spatially close loops, simultaneous closure invariably leads to more accurate predictions than sequential closure. The accuracy of the predictions obtained for pairs of loops in the size range of 5–7 residues each is comparable to that obtained by other methods, when predicting the structures of single loops: the RMS deviations from the native conformations of various test cases modeled are approximately 0.6–1.7 Å for backbone atoms and 1.1–3.3 Å for all‐atoms.
Transmembrane helices predicted at 95% accuracy
Authors: Rost Burkhard , Sander Chris , Casadio Rita , Fariselli Piero 
Published: June 2010

AbstractWe describe a neural network system that predicts the locations of transmembrane helices in integral membrane proteins. By using evolutionary information as input to the network system, the method significantly improved on a previously published neural network prediction method that had been based on single sequence information. The input data were derived from multiple alignments for each position in a window of 13 adjacent residues: amino acid frequency, conservation weights, number of insertions and deletions, and position of the window with respect to the ends of the protein chain. Additional input was the amino acid composition and length of the whole protein. A rigorous cross‐validation test on 69 proteins with experimentally determined locations of transmembrane segments yielded an overall two‐state per‐residue accuracy of 95%. About 94% of all segments were predicted correctly. When applied to known globular proteins as a negative control, the network system incorrectly predicted fewer than 5% of globular proteins as having transmembrane helices. The method was applied to all 269 open reading frames from the complete yeast VIII chromosome. For 59 of these, at least two transmembrane helices were predicted. Thus, the prediction is that about one‐fourth of all proteins from yeast VIII contain one transmembrane helix, and some 20%, more than one.
Principles of protein folding — A perspective from simple exact models
Authors: Dill Ken A. , Bromberg Sarina , Yue Kaizhi , Chan Hue Sun , Ftebig Klaus M. , Yee David P. , Thomas Paul D. 
Published: June 2010

AbstractGeneral principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two‐state cooperativity, secondary and tertiary structures, and multistage folding kinetics —fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating “foldable” chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent‐averse.
Proline cis‐trans isomerization in staphylococcal nuclease: Multi‐substate free energy perturbation calculations
Authors: Hodel Alec , Rice Luke M. , Simonson Thomas , Fox Robert O. , Brünger Axel T. 
Published: June 2010

AbstractStaphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116‐Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (δDLGcis‐trans ≈ 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (ΔGcis‐trans (ΔTScis‐trans ≈ 10 kcal/mol). There is evidence from X‐ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112–117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substates. By partitioning the search space into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X‐ray structures of nuclease variants and yield a model of the unknown minor trans conformation.
NMR studies of novel inhibitors bound to farnesyl‐protein transferase
Authors: Koblan Kenneth S. , Mosser Scott D. , Omer Charles A. , Christopher Culberson J. , Jane Desolms S. , Giuliani Elizabeth A. , Pitzenberger Steven M. , Bogusky Michael J. 
Published: June 2010

AbstractFarnesyl‐protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the carboxyl‐terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for the membrane association and cell‐transforming activities of ras. Inhibitors of FPTase have been demonstrated to inhibit ras‐dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers. The FPTase‐bound conformation of a tetrapeptide inhibitor, CVWM, and a novel pseudopeptide inhibitor, L‐739,787, have been determined by NMR spectroscopy. Distance constraints were derived from two‐dimensional transferred nuclear Overhauser effect experiments. Ligand competition experiments identified the NOEs that originate from the active‐site conformation. Structures were calculated with the combination of distance geometry and restrained energy minimization. Both peptide backbones are shown to adopt nonideal reverse‐turn conformations most closely approximating a type III β‐turn. These results provide a basis for understanding the spatial arrangements necessary for inhibitor binding and selectivity and may aid in the design of therapeutic agents.
Carbohydrate binding sites in a pancreatic α‐amylase‐substrate complex, derived from X‐ray structure analysis at 2.1 Å resolution
Authors: Qian Minxie , Haser Richard , Payan FranÇoise 
Published: June 2010

AbstractThe X‐ray structure analysis of a crystal of pig pancreatic α‐amylase (PPA, EC 3.2.1.1.) that was soaked with the substrate maltopentaose showed electron density corresponding to two independent carbohydrate recognition sites on the surface of the molecule. Both binding sites are distinct from the active site described in detail in our previous high‐resolution study of a complex between PPA and a carbohydrate inhibitor (Qian M, Buisson G, Duée E, Haser H, Payan F, 1994, Biochemistry 55:6284–6294). One of the binding sites previously identified in a 5‐Å‐resolution electron density map, lies at a distance of 20 A from the active site cleft and can accommodate two glucose units. The second affinity site for sugar units is located close to the calcium binding site. The crystal structure of the maltopentaose complex was refined at 2.1 A resolution, to an R‐factor of 17.5%, with an RMS deviation in bond distances of 0.007 Å. The model includes all 496 residues of the enzyme, 1 calcium ion, 1 chloride ion, 425 water molecules, and 3 bound sugar rings. The binding sites are characterized and described in detail. The present complex structure provides the evidence of an increased stability of the structure upon interaction with the substrate and allows identification of an N‐terminal pyrrolidonecarboxylic acid in PPA.