bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparative molecular model building of two serine proteinases from cytotoxic T lymphocytes
Authors: Murphy Michael E. P. , Moult John , Bleackley R. Chris , Gershenfeld Howard , Weissman Irving L. , James Michael N. G. 
Published: May 2005

AbstractTwo genes that are expressed when precursor cytotoxic T lymphocytes are transformed to T killer cells have been cloned and sequenced. The derived amino acid sequences, coding for the cytotoxic cell protease 1 (CCP1) and Hannuka factor (HF) are highly homologous to members of the serine proteinase family. Comparative molecular model building using the known three‐dimensional structures and the derived amino acid sequences of the lymphocyte enzymes has been provided useful information, especially in predicting the conformations of the substrate binding sites. In applying this modelling procedure, we used the X‐ray structures of four serine proteinase to provide a structurally based sequences alignment: α‐chymotrypsin (CHT), bovine trypsin (BT), Streptomyces griseus trypsin (SGT), and rat must cell protease 2 (RMCP2). The root mean square differences in α‐carbon atom positions among these four structures when compared in a pairwise fashions range form 0.79 to 0.97 Å for structurally equivalent residues. Te sequences of the two lymphocyte enzymes were then aligned to these proteinase using chemical criteria and the superimposed X‐ray structures as guides. The alignment showed that the sequence of CCP1 was most similar to RMCP2, whereas HF has regions of homology with both RMCP2 and BT. RmCP2 and BT as templates for HF, the molecular models were constructed. Intermolecular steric clashes that resulted from replacement of amino acid chains of the templates by the aligned residues of CCP1 and HF were relieved by adjustment of the side chain conformational angels in an interactive computer graphics device. This process was followed by energy minimization of the enzyme model to optimize the stereochemical geometry and to relieve any remaining unacceptably close nonbonded contacts. The resulting model of CCP1 has an arginie residue at position 226 in the specificity pocket, thereby predicting a substrate preference for P1 aspartate or glutamate residues. The model also predicts favorable binding for a small hydrophobic residue at the P2 position of the substrate. The primary specificity pocket of HF resembles that of BT and therefore predicts a lysine or arginine preference for the P1 residue. The arginine at position 99 in the model of HF suggests a preference for aspartate or glutamate side chains in the P2 positions of the substrate. Both CCP1 and HF have a free cysteine in the segment of the polypeptide 88 to 93. Models of the dimeric form of these enzymes can be constructed by forming disulfide bridges between the suitably oriented monomers, ie., Cys88–Cys88′ for CCP1 and Cys93–Cys93′ for HF.
Refined structure of human carbonic anhydrase II at 2.0 Å resolution
Authors: Eriksson A. Elisabeth , Jones T. Alwyn , Liljas Anders 
Published: May 2005

AbstractThe structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure–factor least‐squares refinement at 2.0 Å resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stabilize secondary structure elements. The zinc ion is ligated to three histidyl residues and one water molecule in a nearly tetrahedral geometry. In addition to the zinc‐bound water, seven more water molecules are identified in the active site. Assuming that Glu‐106 is deprotonated at pH 8.5, some of the hydrogen bond donor–acceptor relations in the active site can be assigned and are described here in detail. The Oγ1 atom of Thr‐199 donates its proton to the Oε1 atom of Glu‐106 and can function as a hydrogen bond acceptor only in additional hydrogen bonds.
Thermitase, a thermostable subtilisin: Comparison of predicted and experimental structures and the molecular cause of thermostability
Authors: Frömmel Cornelius , Sander Chris 
Published: May 2005

AbstractThe Subtilisin family of proteases has four members of known sequence and structure: subtilisin Carlsberg, Subtilisin novo, proteinase K, and thermitase. Using thermitase as a test case, we ask two questions. How good are methods for model building a three‐dimensional structure of a protein based on sequence homology to a known structure? And what are the molecular causes of thermostability? First, we compare predicted models of thermitase, refined by energy minimization and varied by molecular dynamics, with the preliminary crystal structure. The predictions work best in the conserve structural core and less well in seven loop regions involving insertions and deletions relative to Subtilisin. Here, variation of loop regions by molecular dynamics simulation in vacuo followed by energy minimization does not improve the prediction since we find no correlation between in vacuo energy and correctness of structure when comparing local energy minima. Second, in order to identify the molecular case of thermostability we confront hypotheses erived by calculation of the details of interatomic interactions with inactivation experiments. As a result, we can exclude salt bridges and hydrophobic interactions as main cause of thermostability. Based on a combination of theoretical and experimental evidence, the unusually tight binding of calcium by thermitase emerges as the most likely single influence responsible for its increased thermostability.
Structural principles of α/β barrel proteins: The packing of the interior of the sheet
Authors: Lesk Arthur M. , Brändén Carl‐Ivar , Chothia Cyrus 
Published: May 2005

Abstractα/β barrel structures very similar to that first observed in triose phosphate isomerase are now known to occur in 14 enzymes. To understand the origin of this fold, we analyzed in three of these proteins the geometry of the eight‐stranded β‐sheets and the packing of the residues at the center of the barrel. The Packingin thisregion is seen in its simplest form in glycolate oxidase. It consists of 12 residues arranged in three layers. Each layer contains four side chains. The packing of RubisCO and TIM can be understood in terms of distortions of this simple pattern, caused by residues with small side chains at someof the positions inside the barrel. Two classes of packing are found. In one class, to which RubisCO and TIM belong, the central layer is formed by a residue from the first, third, fifth, and seventh strands; the upper and lower layers are formed by residues fromthe second, fourth, sixth, and eighth strands. In the second class, to which GAO belongs, this is reversed: it is side chains from the even‐numbered strands that form the central layer, and side chains from the oddnumbered strands that form the outer layers. Our results suggest that not all proteins with this fold are related by evolution, but that they represent a common favorable solution to the structural problems involved in the creation of a closed β barrel.
Rapid calculation of the solution scattering profile from a macromolecule of known structure
Authors: Lattman Eaton Edward 
Published: May 2005

AbstractIf one expands the structure factor equation in spherical coordinates, rotational averaging of the molecular Fourier transform, which leads directly to the solution scattering profile, is greatly simplified. It becomes a projection in the polar and azimuthal angular variables. The profile is given by

equation image

The index j runs over all atoms; r, θ, ϕ are atomic coordinates and ε and N are constants; the Ym,n are complex spherical harmonics, and Jn are spherical Bessel functions; R = 2 sin θ/λ. The effects of solvent have been modeled by subtracting from each protein atom a properly weighted water. Hydrogens have been included by using scattering curves fj derived from the spherical averaging ofprotein atoms with their attached hydrogens. This approach may also be satisfactory for neutron scattering. Published scattering profiles2 for lysozyme and BPTI have been accurately matched in less than one‐tenth the time required by other methods. Separate, adjustable temperature factors for the protein, solvent waters, and bound watersare used, and appear to be needed. In the case of BPTI, as suggested by NMR observations, the observed diffraction pattern was much better accounted for by including only 4 tightly bound waters rather than the roughly 60 seen by crystallography.
Secondary structural analysis of retrovirus integrase: Characterization by circular dichroism and empirical prediction methods
Authors: Lin Thy‐Hou , Quinn Thomas P. , Grandgenett Duane , Walsh Mary T. 
Published: May 2005

AbstractThe retrovirus integrase (IN) protein is essentialfor integration of viral DNA into host DNA. The secondary structure of thepurified IN protein from avian myeloblastosis virus was investigated by bothcircular dichroism (CD) spectroscopy and five empirical prediction methods. The secondary structures determined from the resolving of CD spectra through a least‐squares curve fitting procedure were compared with those predicted from four statistical methods, e.g., the Chou–Fasman, arnier–Osguthorpe–Robson, Nishikawa–Ooi, and a JOINT scheme which combined all three of these methods, plus a pure a priori one, the Ptitsyn–Finkelstein method. Among all of the methods used, the Nishikawa–Ooiprediction gave the closest match in the composition of secondary structureto the CD result, although the other methods each correctly predictedoneor more secondary structural group. Most of the α‐helix and β‐sheet states predicted by the Ptitsyn–Finkelstein methodwere in accord with the Nishikawa–Ooi method. Secondary structural predictions by the Nishikawa–Ooi method were extended further toinclude IN proteins from four phylogenetic distinct retroviruses. The structuralrelationships between the four most conserved amino acid blocks of these IN proteins were compared using sequence homology and secondary structure predictions.
The crystal structure of the ternary complex of staphylococcal nuclease, Ca2+ and the inhibitor pdTp, refined at 1.65 Å
Authors: Loll Patrick J. , Lattman Eaton E. 
Published: May 2005

AbstractThe structure of a complex of staphylococcal nuclease with Ca2+ and deoxythymidine 3′,5′‐biophosphate (pdTp) has been refined by stereochemically restrained leastsquares minimization to a crystallographic R value of 0.161 at Å resolution. The estimated root‐mean‐square (rms) error in the coordinates in 0.16 Å. The final model comprises 1082 protein atoms, onecalcium ion, the pdTp molecule, and 82 protein atoms, onecalcium ion, the pdTp molecule, and 82 solvent water molecules;it displays an rms deviation from ideality of 0.017 Å for bond distances and 1.8° for bond angles.The mean distance between corresponding α carbons in the refined and unrefined structures is 0.6 Å we observe small but significant differences between the refined and unrefined models in the turn between residues 27 and 30, the loop between residues 44 and 50, the first helix, and the extended strand between residues 112 and 117 which forms part of the active site binding pocket.The details of the calcium liganding and solvent structure in the activesite are clearly shown in the final electron density map. The structure ofthe catalytic site is consistent with mechanism that has been proposed for this enzyme. However, we note that two lysines from a symmetry‐related molecule in the crystal lattice may play an important role in determining the geometry of inhibitor binding, and that only one of the two required calcium ions is observed in the crystal structure; thus, caution is advised in extrapolating from the structure of the complex of enzyme and inhibitor to that enzyme and substrate.
PKB: A program system and data base for analysis of protein structure
Authors: Bryant Stephen H. 
Published: May 2005

AbstractPKB is a computer program system that combines a data base of three‐dimensional protein structures with a series of algorithms for pattern recognition, data analysis, and graphics. By typing relatively simple commands the user may search the data base for instances of a structural motif and analyze in detail the set of individual structures that are found. The application of PKB to the study of protein folding is illustrated in three examples. The first analysis compares the conformations observed for a short sequential motif, sequences similar to the cell‐attachment signal Arg‐Gly‐Asp. The second compares sequences observed for a conformational motif, a 16‐residue βαβ unit. The third analysis considers a population of substructures containing ion‐pair interaction, examining the relationship offrequency of occurrence to calculated electrostatic energy.
The structure of aconitase
Authors: Robbins A. H. , Stout C. D. 
Published: May 2005

AbstractThe crystal structure of the 80,000 Da FeS enzyme aconitase has been solved and refined at 2.1 Å resolution. The protein contains four domains; the first three from the N‐terminus are closely associated around the [3Fe–4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Associationof the larger C‐terminal domain with the first three domains createsan extensive cleft leading to the FeS cluster. Residues from all four domains contribute to the active site region, which is defined by the FeS cluster and a bound SOion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well asseveral bound water molecules. Three of these side chains reside on a threeturn 310 helix in the first domain. The SOion is bound 9.3 Å from the center of the [3Fe–4S] cluster by the side chains of 2 Arg and 1 Gln rsidues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.
Evolution of CuZn superoxide dismutase and the Greek Key β‐barrel structural motif
Authors: Getzoff Elizabeth D. , Tainer John A. , Stempien Michelle M. , Bell Graeme I. , Hallewell Robert A. 
Published: May 2005

AbstractDetailed analysis of the CuZn superoxide dismutase (SOD) structure provides new results concerning the significance and molecular basis for sequence conservation, intron‐exon boundary locations, gene duplication, and Greek key β‐barrel evolution. Using 15 aligned sequences, including a new mouse sequence, specific roles have been assigned to all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of uperoxide dismutation. The β‐strands have no sequence insertions and deletions, whereas insertions occur within the loops connecting the β‐strands and at both termini. Thus, the β‐barrel with only four invariant residues is apparently over determined, but dependent on multiple cooperative side chain interactions. The regions encoded by exon I, a proposed nucleation site for protein folding, and exon III, the Zn loop involved in stability and catalysis, are the major structural subdomains not included in the internal twofold axis of symmetry passing near the catalytic Cu ion. This provides strong confirmatory evidence for gene evolution by duplication and fusion followed by the addition of these two exons. The proposed evolutionary pathway explains the structural versatility ofthe Greek key β‐barrel through functional specialization and subdomain insertions in new loop connections, and provides a rationale for the size of the present day enzyme.
Molecular dynamics simulations of ribonuclease T1: Comparison of the free enzyme and 2′ GMP–enzyme complex
Authors: MacKerell Alexander D , Nilsson Lennart , Rigler Rudolf , Heinemann Udo , Saenger Wolfram 
Published: May 2005

AbstractMolecular dynamics simulations were performed on free RNase T1 and the 2′GMP–RNase T1 complex in vacuum and with water in the active site along with crystallographically identified waters, allowing analysis of both active site and overall structural and dynamics changes due to the presence of 2′GMP. Difference in the active site include a closing in the presence of 2′GMP, which is accompanied by a decrease in mobility of active site residues. The functional relevance of the active site fluctuations is discussed. 2′GMP alters the motion of Tyr‐45, suggesting a role for that residue in providing a hydrophobic environment for the protein–nucleic acid interactions responsible for the specificity of RNase T1. The presence of 2′GMP causes a structural change of the C‐terminus of the α‐helix, indicating the transmission of structural changes from the active site through the protein matrix. Overall fluctuations of both the free and 2′GMP enzyme forms are in good agreement with X‐ray temperature factors. The motion of Trp‐59 is influenced by 2′GMP, indicating difference in enzyme dynamics away from the active site, with the calculated changes following those previously seen in time‐resolved fluorescence experiments.
The effects of truncating long‐range forces on protein dynamics
Authors: Loncharich Richard J , Brooks Bernard R 
Published: May 2005

AbstractThis paper considers the effects of truncating long‐range forces on protein dynamics. Six methods of truncation that we investigate as a function of cutoff criterion of the long‐range potentials are (1) a shifted potential; (2) a switching function; (3) simple atom–atom truncation based on distance; (4) simple atom–atom truncation based on a list which is updated periodically (every 25 steps); (5) simple group–group truncation based on distance; and (6) simple group–group truncation based on a list which is updated periodically (every 25 steps). Based on 70 calculations of carboxymyoglobin we show that the method and distance of long range cutoff have a dramatic effect on overall protein behavior. Evaluation of the different methods is based on comparison of a simulation's rms fluctuation about the average coordinates of a no cutoff simulation and from the X‐ray structure of the protein. The simulations in which long‐range forces are truncated by a shifted potential shows large rms deviations for cutoff criteria less than 14 Å, and reasonable deviations and fluctuations at this cutoff distance or larger. Simulations using a switching function are investigated by varying the range over which electrostatic interactions are switched off. Results using a short switching function that switches off the potential over a short range of distances are poor for all cutoff distances. A switching function over a 5–9 Å range gives reasonable results for a distance‐dependent dielectric, but not using a constant dielectric. Both the atom–atom and group–group truncation methods based on distance shows large rms deviations and fluctuation for short cutoff distance, while for cutoff distance of 11 Å or greater, reasonable results are achieved. Although comparison of these to distance‐based truncation methods show surprisingly larger rms deviations for the group–group truncation, contrary to simulation studies of aqueous ionic solutions. The results of atom–atom or group–group list‐based simulations generally appear to be less stable than the distance‐based simulations, and require more frequent velocity scaling or stronger coupling to a heat bath.
Salt or ion bridges in biological system: A study employing quantum and molecular mechanics
Authors: Deerfield David W. , Nicholas Hugh B. , Hiskey Richard G. , Pedersen Lee G. 
Published: May 2005

AbstractEquilibrium geometries and binding energies of model “salt” or “ion” bridge systems have been computed by ab initio quantum chemistry techniques (GAUSSIAN82) and by empirical techniques (AMBER2.0). Formate and dimethyl phosphate served as anions in the model compounds while interacting with several organic cations, including methyl ammonium, methyl guanidinium, and divalent metal ion (either Mg2+ or Ca2+) without and with an additional chloride; and a divalent metal ion (either Mg2+ or Ca2+), chloride, and four water molecules of hydration about the metal ion. The majority of the quantum chemical computations were performed using a split‐valence basis set. For the model compounds studied we find that the ab initio geometries are in remarkably goodagreement with the molecular mechanics geometries.Several Calculations werealso performed using diffuse fractions. The formate anion binds these modelcations more strongly than does dimethyl phosphate, while the organiccation methyl ammonium binds model anions more strongly than does methyl guanidinium. Finally, in model compounds including organic anions, Mg2+ or Ca2+ and four molecules of water, and a chloride anion, we find that the equilibrium structure of the magnesium complex involves a solvent separated ion pair (the magnesium ion is six coordinate), whereas the calcium ion complex remains seven coordinate. Molecular mechanics overestimates binding energies, but the estimates may be close enough to actual binding energies togive useful insight into the details energies to give useful insight into the details of salt bridges in biological systems.
Peptide sequencing and site‐directed mutagenesis identify tyrosine‐319 as the active site tyrosine of Escherichia coli DNA topoisomerase I
Authors: Lynn Richard M. , Wang James C. 
Published: May 2005

AbstractTyrosine 319 of E. coli topoisomerase I is shown to be the activesite tyrosine that becomes covalently attached to a DNA 5′ phosphoryl group during the transient breakage of a DNA internucleotide bond by the enzyme. The tyrosine was mapped by trapping the covalent complex between the DNA and DNA topoisomerase I, digesting the complex exhaustively with trypsin, and sequencing the DNA‐linked tryptic peptide. Site‐directed mutagenesis converting Tyr‐319 to a serine or phenylalanine completely inactivates the enzyme. The structure of the enzyme andits catalysis of DNA strand breakage, passage, and rejoining are discussed in terms of the available information.
Engineering subtilisin BPN′ for site‐specific proteolysis
Authors: Carter Paul , Nilsson Björn , Burnier John P. , Burdick Daniel , Wells James A. 
Published: May 2005

AbstractA combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN′, which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wildtype subtilisin BPN′ is greatly restricted by substitution of the catalytic histidine 64 with alanine (H64A) so that certain histidine‐containing substrates are preferentially hydrolysed (Carter, P., Wells, J. A. Science 237:394–399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20‐fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild‐type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N‐terminal sequence. Several features of H64A variants of subtilisin make them attractive for site‐specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N‐terminal side of the cleavage site and yet are broadly specific on the C‐terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.