bluuebunny/crossref_metadata_2025 huggingface dataset:
Fuzzy cluster analysis of molecular dynamics trajectories
Authors: Gordon Heather L. , Somorjai Rajmund L.
Published: May 2005

We propose fuzzy clustering as a method to analyze molecular dynamics (MD) trajectories, especially of proteins and polypeptides. A fuzzy cluster analysis locates classes of similar three‐dimensional conformations explored during a molecular dynamics simulation. The method can be readily applied to results from both equilibrium and nonequilibrium simulations, with clustering on either global or local structural parameters. The potential of this technique is illustrated by results from fuzzy cluster analyses of trajectories from MD simulations of various fragments of human parathyroid hormone (PTH). For large molecules, it is more efficient to analyze the clustering of root‐mean‐square distances between conformations comprising the trajectory. We found that the results of the clustering analysis were unambiguous, in terms of the optimal number of clusters of conformations, for the majority of the trajectories examined. The conformation closest to the cluster center can be chosen as being representative of the class of structures making up the cluster, and can be further analyzed, for example, in terms of its secondary structure. The CPU time used by the cluster analysis was negligible compared to the MD simulation time. © 1992 Wiley‐Liss, Inc.
A model of the platelet factor 4 complex with heparin
Authors: Stuckey Jeanne A. , Charles Robert St. , Edwards Brian F. P.
Published: May 2005

A model of heparin bound to bovine platelet factor 4 (BPF4) was completed using a graphically designed heparin molecule and the crystallographic coordinates of the native bovine platelet factor 4 tetramer. The oligosaccharides had a chain length of at least eight disaccharide units with the major repeating disaccharide unit consisting of (I→4)‐O‐(α‐L‐idopyranosyluronic acid 2‐sulfate)‐(l→4)‐(2‐deoxy‐2‐sulfamino‐2‐D‐glucopyra‐nosyl 6‐sulfate). Each disaccharide unit carried a −4.0 charge. The structure of BPF4 was solved to 2.6 Å resolution with R = 0.237. Each monomer of BPF4 contains an α‐helix lying across 3 strands of antiparallel β‐sheet. Each helix has four lysines, which have been implicated in heparin binding. These lysine residues are predominantly on one side of the helix and are solvent accessible. Electrostatic calculations performed on the BPF4 tetramer show a ring of strong, positive charge which runs perpendicularly across the helices. Included in this ring of density is His‐38, which has been shown by NMR to have a large pKa shift when heparin binds to BPF4. Our model of heparin bound to PF4 has the anionic polysaccharide perpendicular to the α‐helices, wrapped about the tetramer along the ring of positive charge, and salt linked to all four lysines on the helix of each monomer. © 1992 Wiley‐Liss, Inc.
Regulation of oxygen affinity by quaternary enhancement: Does hemoglobin ypsilanti represent an allosteric intermediate?
Authors: Doyle Michael L. , Lew George , Turner George J. , Rucknagel Donald , Ackers Gary K.
Published: May 2005

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (β99 Asp→xsTyr) have revealed a previously unknownquaternary structure called “quaternary Y” and suggested that the new structure may represent an important intermediate in the cooperative oxygenationpathway of normal hemoglobin.15 Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additionalβ99 mutants (Asp β99→Val, Gly, Asn, Ala, His) totest for consistency between their energetics and those of the intermediatespecies of normal hemoglobin.

Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that thetetramer oxygenation affinity is 85‐fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads toa noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer‐tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation‐dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the α1β2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen. © 1992 Wiley‐Liss, Inc.
Crystal structure of human immunoglobulin fragment Fab new refined at 2.0 Å esolution
Authors: Saul Frederick A. , Poljak Roberto J.
Published: May 2005

The three‐dimensional structure of the human immunoglobulin fragment Fab New (IgG1,λ) has been refined to a crystal‐lographic R‐factor of 16.9% to 2Å resolution. Rms deviations of the final model from ideal geometry are 0.014 Å for bond distances and 3.03° for bond angles. Refinement was based on a new X‐ray data set including 28,301 reflections with F>2.5σ(F) from 6.0 to 2.0 Å resolution. The starting model for the refinement procedure reported here is from the Brookhaven Protein Data Bank entry 3FAB (rev. 1981). Differences between the initial and final models include modified polypeptide‐chain folding in the third complementarity‐determining region (CDR3) and the third framework region (FR3) of VH and in some exposed loops of CL and CHl. Amino acid sequencechanges were determined at a number of positions by inspection of difference electron density maps. The incorporation of amino acid sequence changes results inan improved VH framework model for the “humanization” of monoclonal antibodies.
Folding protein α‐carbon chains into compact forms by monte carlo methods
Authors: Covell David G.
Published: May 2005

A method is presented for generating folded chains of specific aminoacid sequences on a simple cubic lattice. Monte Carlo simulations are used to transform extended geometries of simplified α‐carbon chainsfor eight small monomeric globular proteins into folded states. Permitted chain transitions are limited to a few types of moves, all restricted to occur on the lattice. Crude residue–residue potentials derived from statistical structure data are used to describe the energies for each conformer. The low resolution structures obtained by this procedure contain many of the correct gross features of the native folded architectures with respect to average residue energy per nonbonded contact, segment density, and location of surface loops and disulfide pairs. Rms deviations between these and the native X‐ray structures and percentage of native long‐range contacts found in these final folded structures are 7.6 ± 0.7 Å and 48 ± 3%, respectively. This procedure can be useful for predicting approximate tertiary interactions from amino acid sequence. © 1992 Wiley‐Liss, Inc.
Generation of a substructure library for the description and classification of protein secondary structure. I. Overview of the methods and results
Authors: Prestrelski Steven J. , Williams Arthur L. , Liebman Michael N.
Published: May 2005

Protein secondary structure has been typically classified into four major classes—α‐helices, extended strands, reverse turns, and loops. Available methods for secondary structure analysis utilize predefined structure templates to search for structural matches among proteins. By this approach a significant portion of a proteins backbone conformation is assigned to one of a limited number of conformations or, if unassigned, to random coil. To expand our ability to describe protein secondary structure, we have developed an algorithm that operates independently of a predefined structure template. The procedure uses two geometric descriptors, the linear distance and the backbone dihedral angle, to represent the conformation form the α‐carbon coordinates. The algorithm functions by searching for conformationally equivalent, contiguous fragments without regard to secondary structural classification and is thus independent of the complexity of the backbone fold. The result is a library of conformationally equivalent structure fragments that exhibit some novel characteristics. The library contains features that reproduce the major secondary structure classes as well as defining conformations previously described only as random or undefined conformations. Additionally, the library defines several subclassifications of β‐strands. We present here a validation of this method and a presentation and discussion of the most significant results. In a second study, we report the results of application of this method to spectra‐structure correlations in Fourier transform infrared spectroscopy. © 1992 Wiley‐Liss, Inc.
MD simulation of subtilisin BPN′ in a crystal environment
Authors: Heiner Andreas P. , Berendsen Herman J. C. , van Gunsteren Wilfred F.
Published: May 2005

In this paper we present a molecular dynamics (MD) simulation of subtilisin BPN′ in a crystalline environment containing four protein molecules and solvent. Con‐formational and dynamic properties of the molecules are compared with each other and with respect to the X‐ray structure to test the validity of the force field. The agreement between simulated and experimental structure using the GROMOS force field is better than that obtained in the literature using other force fields for protein crystals. The overall shape of the molecule is well preserved, as is the conformation of α‐helices and β‐strands. Structural differences are mainly found in loop regions. Solvent networks found in the X‐ray structure were reproduced by the simulation, which was unbiased with respect to the crystalline hydration structure. These networks seem to play an important role in the stability of the protein; evidence of this is found in the structure of the active site. The weak ion binding site in the X‐ray structure of subtilisin BPN′ is occupied by a monovalent ion. When a calcium ion is placed in the initial structure, three peptide ligands are replaced by 5 water ligands, whereas a potassium ion retains (in part) its original ligands. Existing force fields yield a reliable method to probe local structure and short‐time dynamics of proteins, providing an accuracy of about 0.1 nm. © 1992 Wiley‐Liss, Inc.
Application of a directed conformational search for generating 3‐D coordinates for protein structures from α‐carbon coordinates
Authors: Bassolino‐Klimas Donna , Bruccoleri Robert E.
Published: May 2005

A directed conformational search algorithm using the program CONGEN (ref. 3), which samples backbone conformers, is described. The search technique uses information from the partially built structures to direct the search process and is tested on the problem of generating a full set of backbone Cartesian coordinates given only α‐carbon coordinates. The method has been tested on six proteins of known structure, varying in size and classification, and was able to generate the original backbone coordinates with RMSs ranging from 0.30–0.87Å for the α‐carbons and 0.5–0.99Å RMSs for the backbone atoms. Cis peptide linkages were also correctly identified. The procedure was also applied to two proteins available with only α‐carbon coordinates in the Brookhaven Protein Data Bank; thioredoxin (SRX) and triacyiglycerol acylhydrolase (TGL). All‐atom models are proposed for the backbone of both these proteins. In addition, the technique was applied to randomized coordinates of flavodoxin to assess the effects of irregularities in the data on the final RMS. This study represents the first time a deterministic conformational search was used on such a large scale. © 1992 Wiley‐Liss, Inc.
Investigation of the function of mutated cellulose‐binding domains of Trichoderma reesei cellobiohydrolase I
Authors: Reinikainen Tapani , Ruohonen Laura , Nevanen Tarja , Laaksonen Leif , Kraulis Per , Jones T. Alwyn , Knowles Jonathan K. C. , Teeri Tuula T.
Published: May 2005

The function of the cellulosebinding domain (CBD) of the cellobiohydrolase I of Trichoderma reesei was studied by site‐directed mutagenesis of two amino acid residues identified by analyzing the 3D structure of this domain. The mutant enzymes were produced in yeast and tested for binding and activity on crystalline cellulose. Mutagenesis of the tyrosine residue (Y492) located at the tip of the wedge‐shaped domain to alanine or aspartate reduced the binding and activity on crystalline cellulose to the level of the core protein lacking the CBD. However, there was no effect on the activity toward small oligosaccharide (4‐methylumbellifery1 β‐D‐lactoside). The mutation tyrosine to histidine (Y492H) lowered but did not destroy the cellulose binding, suggesting that the interaction of the pyranose ring of the substrate with an aromatic side chain is important. However, the catalytic activity of this mutant on crystalline cellulose was identical to the other two mutants. The mutation P477R on the edge of the other face of the domain reduces both binding and activity of CBHI. These results support the hypothesis that both surfaces of the CBD are involved in the interaction of the binding domain with crystalline cellulose. © 1992 Wiley‐Liss, Inc.
Modeling the anti‐CEA antibody combining site by homology and conformational search
Authors: Mas Maria T. , Smith Kenneth C. , Yarmush David L. , Aisaka Kazuo , Fine Richard M.
Published: May 2005

A model for an antibody specific for the carcinoembryonic antigen (CEA) has been constructed using a method which combines the concept of canonical structure with conformational search. A conformational search technique is introduced which couples random generation of backbone loop conformations to a simulated annealing method for assigning side chain conformations. This technique was used both to verify conformations selected from the set of known canonical structures and to explore conformations available at the H3 loop in CEA ab initio. Canonical structures are not available for H3 due to its variability in length, sequence, and observed conformation in known antibody structures Analysis of the results of conformational search resulted in three equally probable conformations for H3 loop in CEA. Force field energies, solvation free energies, exposure of charged residues and burial of hydrophobic residues, and packing of hydrophobic residues at the base of the loop were used as selection criteria. The existence of three equally plausible structures may reflect the high degree of flexibility expected for an exposed loop of this length. The nature of the combining site and features which could be important to interaction with antigen are discussed. © 1992 Wiley‐Liss, Inc.
Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library
Authors: O'Neil Karyn T. , Hoess Ronald H. , Jackson Sharon A. , Ramachandran Narayana Swamy , Mousa Shaker A. , DeGrado William F.
Published: May 2005

Methods have recently been developed to present vast libraries of random peptides on the surface of filamentous phage. To introduce a degree of conformational constraint into random peptides, a library of hexapeptides flanked by cysteine residues (capable of forming cyclic disulfides) was constructed. This library was screened using the platelet glycoprotein, IIb/IIIa, which mediates the aggregation of platelets through binding of fibrinogen. A variety of peptides containing the sequence Arg‐Gly‐Asp or Lys‐Gly‐Asp were discovered and synthesized. The cyclic, disulfide bonded forms of the peptides bound IIb/IIIa with dissociation constants in the nanomolar range, while reduced forms or an analogue in which Ser replaced the Cys residues bound considerably less tightly. These results demonstrate the feasibility for introducing conformational constraints into random peptide libraries and also demonstrates the potential for using phage peptide libraries to discover pharmacologically active lead compounds. © 1992 Wiley‐Liss, Inc.
Insertion of peptide chains into lipid membranes: An off‐lattice Monte Carlo dynamics model
Authors: Milik Mariusz , Skolnick Jeffrey
Published: May 2005

A combination of dynamic Monte Carlo simulation techniques with a hydropathy scale method for the prediction of the location of transmembrane fragments in membrane proteins is described. The new hydropathy scale proposed here is based on experimental data for the interactions of tripeptides with phospholipid membranes (Jacobs, R.E., White, S.H. Biochemistry 26:6127–6134, 1987) and the self‐solvation effect in protein systems (Roseman, M.A., J. Mol. Biol. 200:513–522, 1988). The simulations give good predictions both for the state of association and the orientation of the peptide relative to the membrane surface of a number of peptides including Magainin2, M2δ, and melittin. Furthermore, for Pf1 bacterio‐phage coat protein, in accord with experiment, the simulations predict that the C‐terminus forms a transmembrane helix and the N‐terminus forms a helix which is adsorbed on the surface of the bilayer. Finally, the present series of simulations provide a number of insights into the mechanism of insertion of peptides into cell membranes. © 1993 Wiley‐Liss, Inc.
Characterization of proline‐containing α‐helix (helix F model of bacteriorhodopsin) by molecular dynamics studies
Authors: Sankararamakrishnan R. , Vishveshwara Saraswathi
Published: May 2005

Many of the bilayer spanning segments of membrane transport proteins contain proline residues, and most of them are believed to occur in α‐helical form. A proline residue in the middle of an α‐helix is known to produce a bend in the helix, and recent studies have focused on characterizing such a bend at atomic level. In the present case, molecular dynamics (MD) studies are carried out on helix F model of bacteriorhodopsin (BR) Ace‐(Ala)7‐Trp‐(Ala)2‐Tyr‐Pro‐(Ala)2‐Trp‐(Ala)8‐NHMe and compared with Ace‐(Ala)7‐Trp‐(Ala)2‐Tyr‐(Ala)3‐Trp‐(Ala)8‐NHMe in which the proline is replaced by alanine. The bend in the helix is characterized by structural parameters such as kink angle (α), wobble angle (θ), virtual torsion angle (ρ), and the hydrogen bond distance d (Op−3 … Np+1). The average values and the flexibility involved in these parameters are evaluated. The correlation among the bend related parameters are estimated. The equilibrium side chain orientations of tryptophan and tyrosine residues are discussed and compared with those found in the recently proposed model of bacteriorhodopsin. Finally, a detailed characterization of the bend in terms of secondary structures such as αI, αII and goniometric helices are discussed, which can be useful in the interpretation of the experimental results on the secondary structures of membrane proteins involving the proline residue. © 1993 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray diffraction studies of dogfish C‐reactive protein
Authors: Samudzi Cleopas T. , Nguyen Nga Y. , Rubin J. Ronald
Published: May 2005

Crystals of dogfish (Mustelus canis) C‐reactive protein were obtained through vapor phase equilibration using the sitting drop rod technique with ammonium sulfate as the precipitating agent. The space group was determined to be P1 (triclinic lattice) with unit cell dimensions of a = 82.91, b = 92.25 and c = 103.40 Å; α = 83.36°, β = 89.76°, and γ = 81.30°. These crystals diffract to about 2.6 Å resolution and contain two hexamers in the asymmetric unit. © 1993 Wiley‐Liss, Inc.
Structural energetics of peptide recognition: Angiotensin II/antibody binding
Authors: Murphy Kenneth P. , Xie Dong , Garcia K. Christopher , Amzel L. Mario , Freire Ernesto
Published: May 2005

The ability to predict the strength of the association of peptide hormones or other ligands with their protein receptors is of fundamental importance in the fields of protein engineering and rational drug design. To form a tight complex between a flexible peptide hormone and its receptor, the large loss of configurational entropy must be overcome. Recently, the crystallographic structure of the complex between angiotensin II and the Fab fragment of a high affinity monoclonal antibody has been determined (Garcia, K. C., Ronco, P. M., Verroust, P. J., Brünger, A. T., Amzel, L. M. Three‐dimensional structure of an angiotensin II–Fab complex at 3 Å: Hormone recognition by an anti‐idiotypic antibody. Science 257:502–507, 1992). In this paper we present a study of the thermodynamics of the association by high sensitivity isothermal titration calorimetry. The results of the experiments indicate that at 30°C the binding is characterized by (1) a ΔH of −8.9 ± 0.7 kcal mol−1, (2) a ΔCp of −240 ± 20 cal K−1 mol−1, and (3) the release of 1.1 ± 0.1 protons per binding site in the pH range 6.0–7.3. Using these values and the previously determined binding constant in phosphate buffer, ΔG at 30°C is estimated as −11 kcal mol−1 and ΔS as 6.9 cal K−1 mol−1. The calorimetric data indicate that binding is favored both enthalpically and entropically. These results have been complemented by structural thermodynamic calculations. The calculated and experimentally determined thermodynamic quantities are in good agreement. Entropically, the loss of configurational entropy is more than compensated by the entropy gain from solvent release associated with the hydrophobic effect. Enthalpically, binding is favored by polar interactions (hydrogen bonding). Consequently, the problem of binding flexible hormones is solved in much the same way as the folding of an unstructured polypeptide chain into a globular protein. © 1993 Wiley‐Liss, Inc.
Successful prediction of the coiled coil geometry of the GCN4 leucine zipper domain by simulated annealing: Comparison to the X‐ray structure
Authors: Nilges Michael , BrüNger Axel T.
Published: May 2005

The recently solved X‐ray structure of the dimerization region (“leucine zipper”) of the yeast transcriptional activator GCN4 (O'Shea, E. K., Klemm, J. D., Kim, P. S., Alber, T. Science 254:539–544, 1991) is compared to previously predicted models which had been obtained by a conformational search procedure employing simulated annealing without any knowledge of the crystal coordinates (Nilges, M., Brünger, A. T. Protein Eng. 4:649–659,1991). During the course of the simulated annealing procedure, the models converged towards the X‐ray structure. The averaged root mean square difference between the models and the X‐ray structure is 1.26 and 1.75 Å for backbone atoms and all nonhydrogen atoms at the dimerization interface, respectively. The local helix–helix crossing angle of the X‐ray structure falls within the range predicted by the models; a slight unwinding of the coiled coil toward the N‐terminal DNA‐binding end of the dimerization region has been correctly predicted. Distance maps between the helices are largely identical. The region around asparagine 20 is asymmetric in the X‐structure and in the models. Surface side chain dihedrals showed a large variation in the models although the χ1, χ2, χ3, χ4 3‐fold dihedrals were correctly predicted in 69, 42, 43, and 44% of the cases, respectively. Phenomenological free energies of dimerization of the models show little correlation with the root mean square difference between the models and the X‐ray structure. © 1993 Wiley‐Liss, Inc.
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

The 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

The 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

The 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

In 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

The 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

We 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

The 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

Dienelactone 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

An 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

Certain 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

Various 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

The 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

Six 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

Enolase 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

An 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.
A molecular dynamics study of solvent behavior around a protein
Authors: Komeiji Yuto , Uebayasi Masami , Someya Jun‐ichiro , Yamato Ichiro
Published: May 2005

The solvent structure and behavior around a protein were examined by analyzing a trajectory of molecular dynamics simulation of thetrp‐holorepressor in a periodic box of water. The calculated selfdiffusion coefficient indicated that the solvent within 10 Å of the protein had lower mobility. Examination of the solvent diffusion around different atoms of different kinds of residues showed no general tendency. Thisfact suggested that the solvent mobility is not influenced significantly bythe kind of the atom or residue they solvated. Distribution analysis aroundthe protein revealed two peaks of water oxygen: a sharp one at 2.8 Å around polar and charged atoms and a broad one at ∼3.4 Å aroundapolar atoms. The former was stabilized by water–protein hydrogen bonds, and the latter was stabilized by water‐lwater hydrogen bonds, suggesting the existence of a hydrophobic shell. An analysis of protein atom–water radial distribution functions confirmed these shell structures around polar or charged atoms and apolar ones. © 1993 Wiley‐Liss, Inc.