bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the backbone dynamics of an anti‐digoxin antibody VL domain by inverse detected 1H–15N NMR: Comparisons with X‐ray data for the Fab
Authors: Constantine Keith L. , Friedrichs Mark S. , Goldfarb Valentina , Jeffrey Philip D. , Sheriff Steven , Mueller Luciano 
Published: May 2005

AbstractThe dynamic behavior of the polypeptide backbone of a recombinant anti‐digoxin antibody VL domain has been characterized by measurements of 15N T1 and T2 relaxation times, 1H–15N NOE values, and 1H–2H exchange rates. These data were acquired with 2D inverse detected heteronuclear 1H–15N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R2 (= 1/T2). All characterized residues display low‐amplitude picosecond timescale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R2 exchange contributions, which reflect motions on the microsecond to millisecond timescale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R2 exchange contributions. The measured 1H–2H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B‐factors derived from X‐ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X‐ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of β‐strands A, B, and G. However, on a residue‐specific level, correlations among the various NMR data, and between the NMR and X‐ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the VL domain, including the three hypervariable loops, undergo dynamic changes upon VL:VH association and/ or complexation with digoxin. Overall, the 26–10 VL domain exhibits relatively low flexibility on the ps–ns timescale. The possible functional consequences of this result are considered. © 1993 Wiley‐Liss, Inc.
Binding interactions of kistrin with platelet glycoprotein IIb‐IIIa: Analysis by site‐directed mutagenesis
Authors: Dennis Mark S. , Carter Paul , Lazarus Robert A. 
Published: May 2005

AbstractThe binding interactions between platelet fibrinogen receptor, glycoprotein (GP) IIb‐IIIa, and kistrin, a snake venom disintegrin protein that contains the adhesion site recognition sequence Arg‐Gly‐Asp (RGD) and potently inhibits platelet aggregation, have been investigated by site‐directed mutagenesis of a synthetic kistrin gene. Kistrin was expressed as a fusion protein in Escherichia coli under control of the alkaline phosphatase promoter. This construction included the stII signal sequence to direct secretion to the periplasmic space and one synthetic (Z) domain of Staphylococcal protein A to allow affinity purification using IgG Sepharose. Kistrin was cleaved from the Z‐domain by site‐specific proteolysis using a mutant subtilisin BPN' and purified by reverse‐phase HPLC. This approach facilitated the rapid purification of a set of 43 alanine replacement mutants whose relative affinity for GP IIb‐IIIa was measured by competition with immobilized kistrin and by inhibition of platelet aggregation in human platelet‐rich plasma. Alanine replacements at R49, G50, and D51 led to weaker inhibitors of platelet aggregation by 90‐fold, 2‐fold, and >200‐fold, respectively. The conservative D51E mutant was still >100‐fold less potent whereas R49K had a minor effect (1.8‐fold), implying the critical nature of the aspartate for high affinity binding. However, mutations outside of the RGD region led to proteins indistinguishable from kistrin, suggesting no substantial secondary binding interactions. Furthermore, reduced kistrin is not active. We therefore propose that a favorable conformation of the RGD region alone is responsible for the high affinity binding of kistrin to GP IIb‐IIIa. © 1993 Wiley‐Liss, Inc.
Crystallographic analysis of the interaction between cyclosporin A and the Fab fragment of a monoclonal antibody
Authors: Vix Olivier , Rees Bernard , Thierry Jean‐Claude , Altschuh Danièle 
Published: May 2005

AbstractThe structure of the complex between cyclosporin A and the Fab fragment of a monoclonal antibody has been established by Crystallographic analysis to 2.65 Åresolution. The structure has been solved by molecular replacement using a composite Fab model. The current R‐factor after refinement is 0.179 between 8 and 2.65Åresolution. The antibody is one among three known structures with long H3 loops. This loop conformation is observed for the first tune in the presence of the antigen. Residues from all six hypervariable loops interact with cyclosporin A. However, the 17 residues long loop H3 is the main contributor to the buried combining site area and to the van der Waals contacts made with cyclosporin A, with 52 and 63%, respectively, of the total contribution. © 1993 Wiley‐Liss, Inc.
Effect of end group blockage on the properties of a class A amphipathic helical peptide
Authors: Venkatachalapathi Y. V. , Phillips Michael C. , Epand Richard M. , Epand Raquel F. , Tytler Ewan M. , Segrest Jere P. , Anantharamaiah G. M. 
Published: May 2005

AbstractIn a recent classification of biologically active amphipathic α‐helixes, the lipid‐associating domains in exchangeable plasma apolipoproteins have been classified as class A amphipathic helixes (Segrest, J. P., De Loof, H., Dohlman, J. G., Brouillette, C. G., Anantharamaiah, G. M. Proteins 8:103–117, 1990). A model peptide analog with the sequence, Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (18A), possesses the characteristics of a class A amphipathic helix. The addition of an acetyl group at the α‐amino terminus and an amide at the α‐carboxyl terminus, to obtain Ac‐18A‐NH2, produces large increases in helicity for the peptide both in solution and when associated with lipid (for 18A vs Ac‐18A‐NH2, from 6 to 38% helix in buffer and from 49 to 92% helix when bound to dimyristoyl phosphatidylcholine in discoidal complexes). Blocking of the end‐groups of 18A stabilizes the α‐helix in the presence of lipid by approximately 1.3 kcal/mol. There is also an increase in the self‐association of the blocked peptide in aqueous solution. The free energy of binding to the PC–water interface is increased only by about 3% (from −8.0 kcal/mol for 18A to −8.3 kcal/mol for Ac‐18A‐NH2). The Ac‐18A‐NH2 has a much greater potency in raising the bilayer to hexagonal phase transition temperature of dipalmitoleoyl phosphatidylethanolamine than does 18A. In this regard Ac‐18A‐NH2 more closely resembles the behavior of the apolipoprotein A‐I, which is the major protein component of high‐density lipoprotein and a potent inhibitor of lipid hexagonal phase formation. The activation of the plasma enzyme lecithin: cholesterol acyltransferase by the Ac‐18A‐NH2 peptide is greater than the 18A analog and comparable to that observed with the apo A‐I. In the case of Ac‐18A‐NH2, the higher activating potency may be due, at least in part, to the ability of the peptide to micellize egg PC vesicles. © 1993 Wiley‐Liss, Inc.
Theoretical probes of conformational fluctuations in S‐peptide and RNase A/3′–UMP enzyme product complex
Authors: Straub John E. , Thirumalai D. 
Published: May 2005

AbstractThe dynamic properties of the RNase A/3′–UMP enzyme/product complex and the S‐peptide of RNase A have been investigated by molecular dynamics simulations using suitable generalization of ideas introduced to probe the energy landscape in structural glasses. We introduce two measures, namely, the kinetic energy fluctuation metric and the force metric, both of which are used to calculate the time needed for sampling the conformation space of the molecules. The calculation of the fluctuation metric requires a single trajectory whereas the force metric is computed using two independent trajectories. The vacuum MD simulations show that for both systems the time required for kinetic energy equipartitioning is surprisingly long even at high temperatures. We show that the force metric is a powerful means of probing the nature and relative importance of conformational substates which determine the dynamics at low temperatures. In particular the time dependence of the non‐bonded force metric is used to demonstrate that at low temperatures the system is predominantly localized hi a single cluster of conformational substates. The force metric is used to show that relaxation of long range (in sequence space) interactions must be mediated by a sequence of local dihedral angle transitions. We also argue that the time needed for compact structure formation is intimately related to the time needed for the relaxation of the dihedral angle degrees of freedom. The tame for non‐bonded interactions, which drive protein molecules to fold under appropriate conditions, to relax becomes extremely long as the temperature is lowered suggesting that the formation of maximally compact structure hi proteins must be a very slow process. © 1993 Wiley‐Liss, Inc.
Packing and recognition of protein structural elements: A new approach applied to the 4‐helix bundle of myohemerythrin
Authors: Tufféry Pierre , Lavery Richard 
Published: May 2005

AbstractWe present a novel search strategy for determining the optimal packing of protein secondary structure elements. The approach is based on conformational energy optimization using a predetermined set of side chain rotamers and appropriate methods for sampling the conformational space of peptide fragments having fixed backbone geometries. An application to the 4‐helix bundle of myohemerythrin is presented. It is shown that the conformations of the amino acid side chains are largely determined at the level of helix pairs and that superposition of these results can be used to construct the full bundle. The final solution obtained, taking into account restrictions due to the lateral amphiphilicity of the helices, differs from the native structure by only a 20° rotation of a single helix. © 1993 Wiley‐Liss, Inc.
Refined structure of bovine carbonic anhydrase III at 2.0 Å resolution
Authors: Eriksson A. Elisabeth , Liljas Anders 
Published: May 2005

AbstractThe three‐dimensional structure of bovine carbonic anhydrase III (BCA III) from red skeletal muscle cells has been determined by molecular replacement methods. The structure has been refined at 2.0 Å resolution by both constrained and restrained structure‐factor least squares refinement. The current crystallographic R‐value is 19.2% and 121 solvent molecules have so far been found associated with the protein. The structure is highly similar to the refined structure of human carbonic anhydrase II. Some differences in amino acid sequence and structure between the two isoenzymes are discussed. In BCA III, Lys 64 and Arg 91 (His 64 and Ile 91 in HCA II) are both pointing out from the active site cavity forming salt bridges with Glu 4 and Asp 72 (His 4 and Asp 72 in HCA II), respectively. However, Arg 67 and Phe 198 (Asn 67 and Leu 198 in HCA II) are oriented towards the zinc ion and significantly reduce the volume of the active site cavity. Phe 198 particularly reduces the size of the substrate binding region at the “deep water” position at the bottom of the cavity and we sugest that this is one of the major reasons for the differences in catalytic properties of isoenzyme III as compared to isozyme II. © 1993 Wiley‐Liss, Inc.
Catalysis by dienelactone hydrolase: A variation on the protease mechanism
Authors: Cheah Eong , Ashley Gary W. , Gary Jon , Oilis David 
Published: May 2005

AbstractDienelactone hydrolase (DLH), an enzyme from the β‐ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg‐81 and Arg‐206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys‐123, His‐202, Asp‐171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley‐Liss, Inc.
Prediction of protein folding class from amino acid composition
Authors: Dubchak Inna , Holbrook Stephen R. , Kim Sung‐Hou 
Published: May 2005

AbstractAn empirical relation between the amino acid composition and three‐dimensional folding pattern of several classes of proteins has been determined. Computer simulated neural networks have been used to assign proteins to one of the following classes based on their amino acid composition and size: (1) 4α‐helical bundles, (2) parallel (α/β)8 barrels, (3) nucleotide binding fold, (4) immunoglobulin fold, or (5) none of these. Networks trained on the known crystal structures as well as sequences of closely related proteins are shown to correctly predict folding classes of proteins not represented in the training set with an average accuracy of 87%. Other folding motifs can easily be added to the prediction scheme once larger databases become available. Analysis of the neural network weights reveals that amino acids favoring prediction of a folding class are usually over represented in that class and amino acids with unfavorable weights are underrepresented in composition. The neural networks utilize combinations of these multiple small variations in amino acid composition in order to make a prediction. The favorably weighted amino acids in a given class also form the most intramolecular interactions with other residues in proteins of that class. A detailed examination of the contacts of these amino acids reveals some general patterns that may help stabilize each folding class. © 1993 Wiley‐Liss, Inc.
Structural energetics of the molten globule state
Authors: Haynie Donald T. , Freire Ernesto 
Published: May 2005

AbstractCertain partly ordered protein conformations, commonly called “moltenglobule states,” are widely believed to represent protein folding intermediates. Recentstructural studies of molten globule states ofdifferent proteins have revealed features whichappear to be general in scope. The emergingconsensus is that these partly ordered forms exhibit a high content of secondary structure, considerable compactness, nonspecific tertiary structure, and significant structural flexibility. These characteristics may be used to define ageneral state of protein folding called “the molten globule state,” which is structurally andthermodynamically distinct from both the native state and the denatured state. Despite exaatensive knowledge of structural features of afew molten globule states, a cogent thermodynamic argument for their stability has not yetbeen advanced. The prevailing opinion of thelast decade was that there is little or no enthalpy difference or heat capacity differencebetween the molten globule state and the unfolded state. This view, however, appears to beat variance with the existing database of protein structural energetics and with recent estimates of the energetics of denaturation of α‐lactalbumin, cytochrome c, apomyoglobin, and T4 lysozyme. We discuss these four proteins at length. The results of structural studies, together with the existing thermodynamic values for fundamental interactions in proteins, provide the foundation for a structural thermodynamic framework which can account for the observed behavior of molten globule states. Within this framework, we analyze the physical basis for both the high stability of several molten globule states and the low probability of other protential folding intermediates. Additionally, we consider, in terms of reduced enthalpy changes and disrupted cooperative interactions, the thermodynamic basis for the apparent absence of a thermally induced, cooperative unfolding transition for some molten globule states. © 1993 Wiley‐Liss, Inc.
Structure determination of feline panleukopenia virus empty particles
Authors: Agbandje Mavis , McKenna Robert , Rossmann Michael G , Strassheim M. Lisa , Parrish Colin R 
Published: May 2005

AbstractVarious crystal forms of the single‐stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P212121, with unit cell dimensions a = 380.1 Å, b = 379.3 Å, and c = 350.9 Å, has been determined to 3.3 Å resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self‐rotation function and their positions were obtained with an R‐factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 Å resolution and gradually extended to 3.3 Å resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA. © Wiley‐Liss, Inc.
Active site dynamics of acyl‐chymotrypsin
Authors: Nakagawa Setsuko , Yu Hsiang‐Ai , Karplus Martin , Urneyama Hideaki 
Published: May 2005

AbstractThe motions of water molecules, the acyl moiety, the catalytic triad, and the oxyanion binding site of acyl‐chymotrypsin were studied by means of a stochastic boundary molecular dynamics simulation. A water molecule that could provide the nucleophilic OH− for the deacylation stage of the catalysis was found to be trapped between the imidazole ring of His‐57 and the carbonyl carbon of the acyl group. It makes a hydrogen bond with the Nε2 of His‐57 and is heldin place through a network of hydrogen‐bonded water molecules in theactive site. The water molecule was found as close as 2.8 Å to the carbonyl carbon. This appears to be due to the constraints imposed by nonbonded interaction in the active site. Configurations were found in which one hydrogen of the trapped water shared a bifurcated hydrogen bond with His‐57‐Nε2 and Ser‐195‐0γ with the water oxygen very close to the carbonyl carbon. The existence of such a water molecule suggests that large movement of the His‐57 imidazole ring between positions suitable for providing general‐base catalyzed assistance and for providing general‐acid catalyzed assistance may notbe required during the reaction. The simulation indicates that the side chains of residues involved in catalysis (i.e., His‐57, Ser‐195, and Asp‐102) are significantly less flexible than other side chains in the protein. The 40% reduction in rms fluctuations is consistent with a comparable reduction calculated from the temperature factors obtained in the X‐ray crystal‐lographic data of γ‐chymotrypsin. The greater rigidity of active site residues seems to result from interconnected hydrogen bonding networks among the residues and between the residues and the solvent water in the active site. © Wiley‐Liss, Inc.
Correlation between protein stability and crystal properties of designed ROP variants
Authors: Kokkinidis Michael , Vlassi Metaxia , Papanikolaou Yannis , Kotsifaki Dina , Kingswell Adrian , Tsernoglou Demetrius , Hinz Hans‐Juuml;rgen 
Published: May 2005

AbstractSix variants of the ROP protein, designed with the aim to analyze by X‐ray crystallography loop formation and core packing interactions in 4‐α‐helical bundles‐ have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolutionX‐ray diffraction studies. For all mutants crystal size‐ sensitivity to X‐irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry‐ in a manner which is not yet understood in detail. © Wiley‐Liss, Inc.
Fluoride inhibition of yeast enolase: Crystal structure of the enolase–Mg2+–F−–Pi complex at 2.6 Å resolution
Authors: Lebioda Lukasz , Zhang Erli , Lewinski Krzysztof , Brewer John M. 
Published: May 2005

AbstractEnolase in the presence of its physiological cofactor Mg2+ is inhibited by fluoride and phosphate ions in a strongly cooperative manner (Nowak, T, Maurer, P. Biochemistry 20:6901, 1981). The structure of the quaternary complex yeast enolase–Mg2+–F−–Pi has been determined by X‐ray diffraction and refined to an R = 16.9% for those data with F/σ(F) ≥ 3 to 2.6 Å resolution with a good geometry of the model. The movable loops of Pro‐35‐Ala‐45, Val‐153‐Phe‐lo9, and Asp‐255‐Asn‐266 are in the closed conformation found previously in the precatalytic substrate–enzyme complex. Calculations of molecular electrostatic potential show that this conformation stabilizes binding of negatively charged ligands at the Mg2+ ion more strongly than the open conformation observed in the native enolase. This closed conformation is complementary to the transition state, which also has a negatively charged ion, hydroxide, at Mg2+. The synergism of inhibition by F− and Pi most probably is due to the requirement of Pi, for the closed conformation. It is possible that other Mg2+‐dependent enzymes that have OH− ions bound to the metalion in the transition state also will be inhibited by fluoride ions. © Wiley‐Liss, Inc.
Principles and pitfalls in designing site‐directed peptide ligands
Authors: Edmundson A. B. , Harris D. L. , Fan Z.‐C. , Guddat L. W. , Schley B. T. , Hanson B. L. , Tribbick G. , Geysen H. M. 
Published: May 2005

AbstractAn immunoglobulin light chain dimer with a large generic binding cavity was used as a host molecule for designing a series of peptide guest ligands. In a screening procedure peptides coupled to solid supports were systematically tested for binding activity by enzyme linked immunosorbent assays (ELISA). Key members of the binding series were synthesized in milligram quantities and diffused into crystals of the host molecule for X‐ray analyses. These peptides were incrementally increased in size and affinity until they nearly filled the cavity. Progressive changes in binding patterns were mapped by comparisons of crystallo‐graphically refined structures of 14 peptide–protein complexes at 2.7 Å resolution. These comparisons led to guidelines for ligand design and also suggested ways to modify previously established binding patterns. By manipulating equilibria involving histidine, for example, it was possible to abolish one important intramolecular interaction of the bound ligand and substitute another. These events triggered a change inconformation of the ligand from a compact to an extended form and a comprehensive change in the mode of binding to the protein. In dipeptides of histidine and proline, protonation of both imidazolium nitrogen atoms was used to program anend‐to‐end reversal of the direction in which the ligand was inserted into the binding cavity. Peptides cocrystallized with proteins produced complexes somewhat different in structure from those in which ligandswere diffused into preexisting crystals. In sucha large and malleable cavity, space utilization was thus different when a ligand was introduced before the imposition of crystal packing restraints. © 1993 Wiley‐Liss, Inc.