bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular mechanics simulations of a conformational rearrangement of D‐xylose in the active site of D‐xylose isomerase
Authors: Smart Oliver S. , Akins John , Blow David M. 
Published: May 2005

AbstractA proposed reaction mechanism for the enzyme D‐xylose isomerase involves the ring opening of the cyclic substrate with a subsequent conformational rearrangement to an extended open‐chain form. Restrained energy minimization was used to simulate the rearrangement. In the ring‐opening step, the substrate energy function was gradually altered from a cyclic to an open‐chain form, with energy minimization after each change. The protein/sugar contact energy did not increase significantly during the process, showing that there was no steric hindrance to ring opening. The conformational rearrangement involves an alteration in the coordination of the substrate to metal ion [1], which was induced by gradually changing restraints on metal/ligand distances. By allowing varying amounts of flexibility in the protein and examining a simplified model system, the interactions of the sugar with metal ion [1] and its immediate ligands were found to be the most important contributors to the energy barrier for the change. Only small changes in the positions of protein atoms were required. The energy barrier to the rearrangement was estimated to be less than the Arrhenius activation energy for the enzymatic reaction. This is in accordance with experimental indications that the isomerization step is rate determining. © 1992 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray diffraction analysis of staphylococcal enterotoxin type C
Authors: Bohach Gregory A. , Chi Young‐In , Stauffacher Cynthia V. 
Published: May 2005

AbstractThe Type C staphylococcal enterotoxin produced by Staphylococcus aureus strain FRI‐909 has been crystallized using a combination of two precipitants, ammonium sulfate and polyethylene glycol 400, with the addition of small amounts of detergent. Two related crystal forms have been obtained, one triclinic, and one tetragonal, both with one toxin molecule per asymmetric unit. These crystals are stable for at least 75 hr in the X‐ray beam and diffract to at least 2.2 and 2.6 Å, respectively. The triclinic crystals have unit cell parameters a = 38.5 Å, b = 43.7 Å, c = 36.9 Å, and interaxial angles α = 99.9°, β = 95.8°, and γ = 98.5°. The tetragonal crystals are of space group P4122 with unit cell parameters a = 43.4 Å and c = 278.0 Å. © 1992 Wiley‐Liss, Inc.
Detection of local structures in reduced unfolded bovine pancreatic trypsin inhibitor
Authors: Amir Dan , Krausz Sara , Haas Elisha 
Published: May 2005

AbstractThe structure of BPTI and reduced BPTI in concentrated guanidinium HCI (GUHCl) in the presence of glycerol has been probed by measurements of dynamic nonradiative excitation energy transfer between probes attached to its amino groups. Inter probe distance distributions were obtained from analysis of donor fluorescence decay curves and used to characterize local structures in unordered states of the protein. Site specifically fluorescently labeled BPTI derivatives (1‐n)BPTI (n = 15, 20, 41, 46) were used, each carrying a 2‐methoxy‐naphthyl‐1‐methylenyl group (MNA) at the N‐terminal amino group of arg1 and 7‐(dimethylamino)‐coumarin‐4‐yl‐acetyl residue (DA‐coum) at one of its ε‐NH2 groups of the lysine side chains. Analysis of donor fluorescence decay kinetics gave the interprobe distance distributions in the native and denatured states.The N‐terminal‐segment, residues 1–15, is in an extended conformation (with an average interprobe distance of 34 ± 2 Å) in the native state. Upon unfolding by reduction with DTT or β‐mercapto ethanol in 6 M GUHCl/glycerol mixture, the conformation of this segment relaxed to a state characterized by a reduced averageinterprobe distance and a larger width of the distances distribution. The average distance between residues 1 and 26, i.e., between the N‐terminus and the turn of the twisted β sheet element (residues 18–35), increased upon unfolding. At −30°C in the above solvent, the distribution between these two sites was probably composed of two conformational subpopulations. About 45 ±20% of the molecules were characterized by a short interprobe distance (like the native state) representing a compact conformation, and 55 ± 20% of the molecules showed large interprobe distances representing an expanded (unfolded) conformation.Thus local structures seem to exist in reduced denatured BPTI even underdenaturing conditions in 6 M GUHCl/glycerol mixtures. Some of those structures are unstable in guanidinium isothiocyanate (GUSCN). The method introduced here is suitable for probing local structures and very long range interactions in unfolded folded proteins and for search for folding initiation sites (FISs) and early folding intermediates. © 1992 Wiley‐Liss, Inc.
Molecular dynamics of HIV‐1 protease
Authors: Harte William E. , Swaminathan S. , Beveridge David L. 
Published: May 2005

AbstractMolecular dynamics simulations have been carried out based on the GROMOS force field on the aspartyl protease (PR) of the human immunodeficiency virus HIV‐1. The principal simulation treats the HIV‐1 PR dimer and 6990 water molecules in a hexagonal prism cell under periodic bundary conditions and was carried out for a trajectory of 100 psec. Corresponding in vacuo simulations, i.e., treating the isolated protein without solovent, were carried out to study the influence of solvent on the simulation. The results indicate that including waters explicitly in the simlation results in a model considerably closer to the crystal structure than when solvent is neglected. Detailed conformational and helicoidal analysis was perfomed on the solvated form to determine the exact nature of the dynamical model and the exact points of agreement and disagreement with the crystal structure. The calculated dynamical model was furthr elucidated by means of studies of the time evolution of the cross‐correlation coefficients for atomic displacements of the atoms comprising the protein backbone. The cross‐correlation analysis revealed significant aspects of structure originating uniquely in the dynamical motions of the molecule. In particular, an unanticipated troughspace, domain‐domain correlation was found between the mobile flap region covering the active site and a remote regions of the structure, which collectively act somewhat like a molecular cantilever. The significance of these results is discussed with respect to the inactivation of the protease by site‐specific mutagenesis, andin the design of inhibitors. © 1992 Wiley‐Liss, Inc.
NMR docking of a substrate into the X‐ray structure of staphylococcal nuclease
Authors: Weber David J. , Gittis Apostolos G. , Mullen Gregory P. , Abeygunawardana Chitrananda , Lattman Eaton E. , Mildvan Albert S. 
Published: May 2005

AbstractThe conformation of the staphylococcal nuclease‐bound metal–dTdA complex, previously determined by NMR methods [Weber, D.J., Mullen, G.P., Mildvan, A.S. (1991) Biochemistry 30:7425–7437] was docked into the X‐ray structure of the enzyme–Ca2+‐3′,5′‐pdTp complex [Loll, P.J., Lattman, E.E. (1989) Proteins: Struct., Funct., Genet. 5:183–201] by superimposing the metal ions, taking into account intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr‐85, Tyr‐113, and Tyr‐115 to proton resonances of the leaving dA moiety of dTdA, and energy minimization to relieve small overlaps. The proton resonances of the Phe, Tyr, and Trp residues of the enzyme in the ternary enzyme–La3+‐dTdA complex were sequence specifically assigned by 2D phase‐sensitive NOESY, with and without deuteration of the aromatic protons of the Tyr residues, and by 2D heteronu‐clear multiple quantum correlation (HMQC) spectroscopy and 3D NOESY‐HMQC spectros‐copy with 15N labeling. While resonances of most Phe, Tyr and Trp residues were unshifted by the substrate dTdA from those found in the enzyme–La3+–3′,5′‐pdTp complex and the enzyme–Ca2+–3′,5′‐pdTp complex, proton resonances of Tyr‐85, Tyr‐113, Tyr‐115, and Phe‐34 were shifted by 0.08 to 0.33 ppm and the 15N resonance of Tyr‐113 was shifted by 2.1 ppm by the presence of substrate. The optimized position of enzyme‐bound dTdA shows the 5′‐dA leaving group to partially overlap the inhibitor, 3′,5′‐pdTp (in the X‐ray structure). Tne 3′‐TMP moiety of dTdA points toward the solvent in a channel defined by Ile‐18, Asp‐19, Thr‐22, Lys‐45, and His‐46. The phosphate of dTdA is coordinated by the metal, and an adjacent inner sphere water ligand is positioned to donate a hydrogen bond to the general base Glu‐43 and to attack the phosphorus with inversion. Arg‐35 and Arg‐87 donate monodentate hydrogen bonds to different phosphate oxygens of dTdA, with Arg‐87 positioned to protonate the leaving 5′‐oxygen of dA, thus clarifying the mechanism of hydrolysis. Model building of an additional 5′‐dGMP onto the 3′‐oxygen of dA placed this third nucleotide onto a surface cleft near residues Glu‐80, Asp‐83, Lys‐84, and Tyr‐115 with its 3′‐OH group accessible to the solvent, thus defining the size of the substrate binding site as accommodating a trinucleotide. © 1992 Wiley‐Liss, Inc.
NMR restraint analysis of transforming growth factor α: A key component for NMR structure refinement
Authors: Brown Frank K. , Hempel Judith C. , Jeffs Peter W. 
Published: May 2005

AbstractStructures of the protein, transforming growth factor α (TGF‐α), have been derived from NMR data using distance geometry and subsequent energy refinement. Analysis of the sequential NOE distance bounds using a template algorithm provides a check for consistency in the calculation of bounds, stereospecific assignment of prochiral centers, and secondary structure assignment. Application of the template algorithm to the long range NOEs found within the NMR data sets collected at pH 6.3 and pH 3.4 is used to assess the confidence levels for the accuracy of the structures obtained from modeling. The method also provides critical insight in differentiating regions of the structure that are well defined from those that are not. Use of the restraint analysis protocol is shown to be a powerful adjunct to currently used methods for the assignment of protein structures from NMR data. © 1992 Wiley‐Liss, Inc.
Alkaline phosphatase‐somatostatin hybrid proteins as probes for somatostatin‐14 receptors
Authors: Langen Hanno T. , Taylor John W. 
Published: May 2005

AbstractBy inserting appropriate peptide ligands into surface loops on globular proteins, we expect to develop probes for the location, accessibility, and steric and electrostatic environment of these ligand‐binding sites on their membrane‐bound receptors. Three residues in a loop on the surface of E. coli alkaline phosphatase were substituted by an 18‐residue peptide containing the receptor‐binding segment of somatostatin‐14 without significantly affecting the catalytic properties of the enzyme. This hybrid protein was then used to investigate the ligand‐binding site of somatostatin receptors. Tryptic cleavage of the hybrid protein within the inserted sequence, and binding of the hybrid protein to antisomatostatin antibodies demonstrated the surface accessibility of the guest peptide. Both the wild‐type enzyme and the hormone‐enzyme hybrid displaced 125I‐labeled somatostatin from rat brain membrane receptors only at high concentrations. How‐ever, chemical cationization of the hybrid protein, which again did not disturb the phosphatase activity, enhanced its receptor‐binding potency to a level only 23 times lower than that of somatostatin itself and 280 times higher than that of the cationized wild‐type protein. This alkaline phosphatase/somatostatin hybrid protein appears, therefore, to be a suitable starting point for the development of probes for the steric and electrostatic environment of the ligand‐binding site of somatostatin receptors. © 1992 Wiley‐Liss, Inc.
Probing β‐lactamase structure and function using random replacement mutagenesis
Authors: Palzkill Timothy , Botstein David 
Published: May 2005

AbstractA new analytical mutagenesis technique is described that involves randomining the DNA sequence of a short stretch of a gene (3–6 codons) and determining the percentage of all possible random sequences that produce a functional protein. A low percentage of functional random sequences in a complete library of random substitutions indicates that the region mutagenized is important for the structure and/or function of the protein. Repeating the mutagenesis over many regions throughout a protein gives a global perspective of which amino acid sequences in a protein are critical. We applied this method to 66 codons of the gene encoding TEM‐1 β‐lactamase in 19 separate experiments. We found that TEM‐1 β‐lactamase is extremely tolerant of amino acid substitutions: on average, 44% of all mutants with random substitutions function and 20% of the substitutions are expressed, secreted, and fold well enough to function at levels similar to those for the wild‐type enzyme. We also found a few exceptional regions where only a few random sequences function. Examination of the X‐ray structures of homologous β‐lactamases indicates that the regions most sensitive to substitution are in the vicinity of the active site pocket or buried in the hydrophobic core of the protein. DNA sequence analysis of functional random sequences has been used to obtain more detailed information about the amino acid sequence requirements for several regions and this information has been compared to sequence conservation among several related β‐lactamases. © 1992 Wiley‐Liss, Inc.
Empirical solvation models in the context of conformational energy searches: Application to bovine pancreatic trypsin inhibitor
Authors: Williams Roger L. , Vila Jorge , Perrot Georges , Scheraga Harold A. 
Published: May 2005

AbstractContinuum solvation models that estimate free energies of solvation as a function of solvent accessible surface area are computationally simple enough to be useful for predicting protein conformation. The behaviour of three such solvation models has been examined by applying them to the minimization of the conformational energy of bovine pancreatic trypsin inhibitor.The model differ only with regard to how the constants of proportionality between free energy and surface area were derived. Each model was derived by fitting to experimentally measured equilibrium solution properties. For two models, the solution property was free energy of hydration. For the thrid, the property was NMR coupling constants. The purpose of this study is to determine the effect of applying these solvation models to the nonequilibrium conformations of a protein arising in the course of global searches for conformational energy minima. Two approaches were used: (1) local energy minimization of an ensemble of conformations similar to the equilibrium conformation and (2) global search trajectories using Monte Carlo plus minimization starting from a single conformation similar to the equilibrium conformation.For the two models derived from free energy measurements, it was found that both the global searches and local minimizations yielded conformations more similar to the X‐ray crystallographic structures than did searches or local minimizations carried out in the absence of a solvation component of the conformational energy. The model derived from NMR coupling constants behaved similarly to the other models in the context of a global search trajectory. For one of the models derived from measured free energies of hydration, it was found that minimization of an ensemble of near‐equilibrium conformations yielded a new ensemble in which the conformation most similar to the X‐ray determined structure PTI4 had the lowest total free energy.Despite the simplicity of the continuum slvation models, the final conformation generated in the trajectories for each of the models exhibited some of the characteristics that have been reported for conformations obtained from molecular dynamics simulations in the presence of a bath of explicit water molecules. They have smaller root mean square (rms) deviations from the experimentally determined conformation, fewer incorrect hydrogen bonds, and slightly larger radii of gyration than do conformations derived from search trajectories carried out in the absence of sovlent. © 1992 Wiley‐Liss, Inc.
Calmodulin binding to α1‐purothionin: Soloution binding and modeling of the complex
Authors: Rao Usha , Teeter Martha M. , Erickson‐Viitanen Susan , DeGrado William F. 
Published: May 2005

AbstractCD and fluorescence spectroscopic measurements show that calmodulin (CaM) binds to purothionins (alpha;1‐purothionin: α1‐PT; β‐purothionin: β‐PT) in 1:1 stoichiometry with an affinity similar to that exhibited with the tightest binding CaM‐binding peptides. Using the available crystal structures of CaM and α1‐PT, a model has been built for the interaction of CaM and α1‐PT and subjected to potential energy minimization. In the model, there is a bend in the central helix of CaM similar to that suggested by Persechini and Kretsinger (J. Card. Pharm. 12:501–512, 1988). α1‐PT fits snugly into the cavity formed by the bent CaM molecule with each of its two helices making apolar interactions with each of the two hydrophobic clefts situated at the terminal domains of CaM. The complex is further stabilized by numerous polar and electrostatic interactions on the rims of the clefts. Our model is compared with two other similar models previously re‐ported for the CaM complexes with other helical peptides and generalizations about the mode of CaM binding to target proteins are made, which have wide relevance to the function of CaM. By analogy, a similar model is pre‐dicted for a CaM–β‐PT complex. © 1992 Wiley‐Liss, Inc.
Crystal structure of the reduced form of p‐ hydroxybenzoate hydroxylase refined at 2.3 Å resolution
Authors: Schreuder Herman A. , van der Laan Jan M. , Swarte Myra B. A. , Kalk Kor H. , Hol Wim G. J. , Drenth Jan 
Published: May 2005

AbstractThe crystal structure of the reduced form of the enzyme p‐hydroxybenzoate hydroxylase from Pseudomonasm fluorescens, complexed with its substrate p‐hydroxybenzoate, has been obtained by protein X‐ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzyme‐substrate complex in deaerated mother liquor containing 300–400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme‐bound FAD by NADPH. This was confirmed by single crystal spectroscopy.X‐ray data to 2.3 Å were collected on oscillation films using a rotating anode generator as an X‐ray source. After data processing and reduction, restrained least squares refinement using the 1.9 Å structure of the oxidized enzyme‐substrate complex as a starting model, yielded a crystallographic R‐factor of 14.8% for 11,394 reflections. The final model of the reduced complex contains 3,098 protein atoms, the FAD molecule, the substrate p‐hydroxybenzoate and 322 solvent molecules.The structures of the oxidized and reduced forms of the enzyme‐substrate complex were found to be very similar. The root‐mean‐square discrepancy for all atoms between both structures was 0.38 Å. The flavin ring is almost completely planar in the final model, although it was allowed to bend or twist during refinement. The observed angle between the benzene and the pyrimidine ring is 2° This value should be compared with observed values of 10° for the oxidized enzyme‐substrate complex and 19° for the enzyme‐product complex. The position of the substrate is virtually unaltered with respect to its position in the oxidized enzyme. No trace of a bound NADP+ or NADPH molecule was found. © 1992 Wiley‐Liss, Inc.
Effects of changing the interaction between subdomains on the thermostability of Bacillus neutral proteases
Authors: Eijsink Vincent G. H. , Vriend Gerrit , van der Vinne Bernard , Hazes Bart , van den Burg Bertus , Venema Gerard 
Published: May 2005

AbstractVariants of the thermolabile neutral protease (Npr) of B. subtilis (Npr‐sub) and the thermostable neutral protease of B. stearothermophilus (Npr‐ste) were produced by means of site‐directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to alter the interaction between the middle and C‐terminal subdomain of these enzymes. In all Nprs a cluster of hydrophobic contacts centered around residue 315 contributes to this interaction. In thermostable Nprs (like Npr‐ste) a 10 residue β‐hairpin, covering the domain interface, makes an additional contribution. The hydrophobic residue at position 315 was replaced by smaller amino acids. In addition, the β‐hairpin was deleted from Npr‐ste and inserted into Npr‐sub. The changes in thermostability observed after these mutations confirmed the importance of the hydrophobic cluster and of the β‐hairpin for the structural integrity of Nprs. Combined mutants showed that the effects of individual mutations affecting the inter action between the subdomains were not additive. The effects on thermostability decreased as the strength of the subdomain interaction increased. The results show that once the subdomain interface is sufficiently stabilized, additional stabilizing mutations at the same interface do not further increase thermostability. The results are interpreted on the basis of a model for the thermal inactivation of neutral proteases, in which it is assumed that inactivation results from the occurrence of local unfolding processes that render these enzymes susceptible to autolysis. © 1992 Wiley‐Liss, Inc.
Hydrogen exchange in native and denatured states of hen egg‐white lysozyme
Authors: Radford Sheena E. , Buck Matthias , Topping Karen D. , Dobson Christopher M. , Evans Philip A. 
Published: May 2005

AbstractThe hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30°C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 105–107 times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35°C, by lowering the stability of the native state through selective cleavage of the Cys‐6–Cys‐127 disulfide crosslink (CM6−127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20‐fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native‐like structure do not persist significantly in the denatured state of CM6−127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69°C, resembles that of the acid‐denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme. © 1992 Wiley‐Liss, Inc.