bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cu(II)‐Binding properties of a cytochrome c with a synthetic metal‐binding site: His‐X3‐His in an α‐helix
Authors: Todd Robert J. , Van Dam Mariana E. , Casimiro Danilo , Haymore Barry L. , Arnold Frances H. 
Published: May 2005

AbstractA metal‐binding site consisting of two histidines positioned His‐X3‐His in an α‐helix has been engineered into the surface of Saccharomyces cerevisiae iso‐1‐cytochrome c. The synthetic metal‐binding cytochrome c retains its biological activity in vivo. Its ability to bind chelated Cu(II) has been characterized by partitioning in aqueous two‐phase polymer systems containing a polymer‐metal complex, Cu(II)IDA‐PEG, and by metal‐affinity chromatography. The stability constant for the complex formed between Cu(II)IDA‐PEG and the cytochrome c His‐X3‐His site is 5.3 × 104 M−1, which corresponds to a chelate effect that contributes 1.5 kcal mol−1 to the binding energy. Incorporation of the His‐X3‐His site yields a synthetic metal‐binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility.
Thermodynamics of ligand binding and denaturation for his64 mutants of tissue plasminogen activator kringle‐2 domain
Authors: Kelley Robert F. , DeVos Abraham M. , Cleary Scott 
Published: May 2005

AbstractThe contribution of His64 to the function and stability of tissue plasminogen activator (t‐PA) kringle‐2 domain (His244 in t‐PA numbering) has been studied by using microcalorimetric methods to compare the ligand binding and thermal denaturation behavior of wild‐type kringle‐2 and mutants having His64 replaced with Tyr or Phe. This site was examined because modeling studies1 suggested that the His64 side chain could play an important role in ligand binding by forming an ion‐pair with the carboxylate of the ligand, L‐lysine. Kringle‐2 domains were expressed by secretion of the 174‐263 portion of t‐PA in E. coli and purified as previously described for the wild‐type domain.2 Both mutant proteins retain affinity for L‐lysine, although reduced three‐ to four‐fold relative to wild‐type, demonstrating that His64 does not interact with the ligand carboxylate through an ion‐pair interaction or by hydrogen bonding. The H64Y substitution does result in an altered specificity of the lysine binding site with the mutant domain having greatest affinity for a ligand of 6.8 Å chain length, whereas the wild‐type domain prefers an 8.8 Å long ligand. For both wild‐type and mutant, the binding of the optimal chain length ligand is dominated by enthalpic effects (ΔH = −6,000 to −7,000 cal/mol) and TΔS accounts for < 15% of ΔG. In addition, the H64Y mutant differs from wild‐type in the effect of ligand α‐amino group modification on binding affinity. Based on examination of the x‐ray structure recently determined for wild‐type kringle‐2, the specificity changes accompanying the H64Y substitution probably result from changes in side chain interactions in the lysine binding site. Thermal denaturation experiments show that the H64Y mutant is also more stable than the wild‐type protein with the difference in stabilization free energy (ΔΔG) equal to 2.7 kcal/mol at 25°C and pH 3. The increased stability of the mutant appears to be related to the difference in hydrophobicity between His and Tyr.
Experimental and theoretical studies of the three‐dimensional structure of human interleukin‐4
Authors: Curtis Benson M. , Presnell Scott R. , Srinivasan Subhashini , Sassenfeld Helmut , Klinke Ralph , Jeffery Eric , Cosman David , March Carl J. , Cohen Fred E. 
Published: May 2005

AbstractThe structure of human interleukin 4 (IL‐4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL‐4 belonged to the all α‐helix class of protein structures. Secondary structure prediction, site‐directed mutagenesis, and CD spectroscopy suggested a predominantly α‐helical structure, consistent with a four‐helix bundle structural motif. A human/mouse IL‐4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL‐4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four‐helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.
Thiol proteases and aldehyde dehydrogenases: Evolution from a common thiolesterase precursor?
Authors: Hempel John , Nicholas Hugh , Jörnvall Hans 
Published: May 2005

AbstractThe C‐terminal 222 residues of human liver aldehyde dehydrogenase can be aligned with the C‐terminal 226 residues of a thiol protease from Dictyostelium discoideum to yield 47 residue identities, including matching active site cysteine residues. A multiple alignment with three more aldehyde dehydrogenases and three more thiol proteases yields three regions with clustered residue similarities. In the tertiary structure of papain, these three regions are in close proximity although widely separated in primary structure, and many conserved residues are located in the active site groove. The three‐dimensional relationships, the common thiol ester mechanisms of the enzymes, the locations of exon boundaries in the dehydrogenase and protease genes, and the conservation of internal salt‐bridging and disulfide‐paired residues in papain, all appear compatible with the hypothesis of an ancestral relationship between thiol proteases and aldehyde dehydrogenases.
FASTRUN: A special purpose, hardwired computer for molecular simulation
Authors: Fine Richard , Dimmler Gerd , Levinthal Cyrus 
Published: May 2005

AbstractWe describe the design, construction, and performance of a special purpose, hardwired accelerator for molecular mechanical calculations called FASTRUN. The processor was designed at Columbia University in 1984, constructed in the Instrumentation Division of Brookhaven National Laboratory, and delivered to Columbia in final form in 1989. It was rendered functional for molecular mechanics in early 1990. Together with its host Star array processor, FASTRUN has a measured performance for molecular dynamics simulations which compares favorably with present day supercomputers. The hardware replication cost of FASTRUN is on the order of $100,000.00
Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons
Authors: Nicholls Anthony , Sharp Kim A. , Honig Barry 
Published: May 2005

AbstractWe demonstrate in this work that the surface tension, water‐organic solvent, transfer‐free energies and the thermodynamics of melting of linear alkanes provide fundamental insights into the nonpolar driving forces for protein folding and protein binding reactions. We first develop a model for the curvature dependence of the hydrophobic effect and find that the macroscopic concept of interfacial free energy is applicable at the molecular level. Application of a well‐known relationship involving surface tension and adhesion energies reveals that dispersion forces play little or no net role in hydrophobic interactions; rather, the standard model of disruption of water structure (entropically driven at 25°C) is correct. The hydrophobic interaction is found, in agreement with the classical picture, to provide a major driving force for protein folding. Analysis of the melting behavior of hydrocarbons reveals that close packing of the protein interior makes only a small free energy contribution to folding because the enthalpic gain resulting from increased dispersion interactions (relative to the liquid) is countered by the freezing of side chain motion. The identical effect should occur in association reactions, which may provide an enormous simplification in the evaluation of binding energies. Protein binding reactions, even between nearly planar or concave/convex interfaces, are found to have effective hydrophobicities considerably smaller than the prediction based on macroscopic surface tension. This is due to the formation of a concave collar region that usually accompanies complex formation. This effect may preclude the formation of complexes between convex surfaces.
Computer design of bioactive molecules: A method for receptor‐based de novo ligand design
Authors: Moon Joseph B. , Howe W. Jeffrey 
Published: May 2005

AbstractThe design of molecules to bind specifically to protein receptors has long been a goal of computer‐assisted molecular design. Given detailed structural knowledge of the target receptor, it should be possible to construct a model of a potential ligand, by algorithmic connection of small molecular fragments, that will exhibit the desired structural and electrostatic complementarity with the receptor. However, progress in this area of receptorbased, de novo ligand design has been hampered by the complexity of the construction process, in which potentially huge numbers of structures must be considered. By limiting the scope of the structure‐space examined to one particular class of ligands–namely, peptides and peptide‐like compounds–the problem complexity has been reduced to the point that successful, de novo design is now possible. The methodology presented employs a large template set of amino acid conformations which are iteratively pieced together in a model of the target receptor. Each stage of ligand growth is evaluated according to a molecular mechanicsbased energy function, which considers van der Waals and coulombic interactions, internal strain energy of the lengtheining ligand, and desolvation of both ligand and receptor. The search space is managed by use of a data tree which is kept under control by pruning according to the energy evaluation. Ligands grown by this procedure are subjected to follow‐up evaluation in which an approximate binding enthalpy is determined. This methodology has proven useful as a precise model‐builder and has also shown the ability to design bioactive ligands.
Subunit assembly and active site location in the structure of glutamate dehydrogenase
Authors: Baker Patrick J. , Britton K. Linda , Engel Paul C. , Farrants George W. , Lilley Kathryn S. , Rice David W. , Stillman Timothy J. 
Published: May 2005

AbstractThe three‐dimensional crystal structure of the NAD+‐linked glutamate dehydrogenase from Clostridium symbiosum has been solved to 1.96 Å resolution by a combination of isomorphous replacement and molecular averaging and refined to a conventional crystallographic R factor of 0.227. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self‐assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical dinucleotide binding fold but with the direction of one of the strands reversed. Difference Fourier analysis on the binary complex of the enzyme with NAD+ shows that the dinucleotide is bound in an extended conformation with the nicotinamide moiety deep in the cleft between the two domains. Hydrogen bonds between the carboxyamide group of the nicotinamide ring and the side chains of T209 and N240, residues conserved in all hexameric GDH sequences, provide a positive selection for the syn conformer of this ring. This results in a molecular arrangement in which the A face of the nicotinamide ring is buried against the enzyme surface and the B face is exposed, adjacent to a striking cluster of conserved residues including K89, K113, and K125. Modeling studies, correlated with chemical modification data, have implicated this region as the glutamate/2‐oxoglutarate binding site and provide an explanation at the molecular level for the B type stereospecificity of the hydride transfer of GDH during the catalytic cycle.
Protein design on computers. Five new proteins: Shpilka, grendel, fingerclasp, leather, and aida
Authors: Sander Chris , Vriend Gerrit , Bazan Fernando , Horovitz Amnon , Nakamura Haruki , Ribas Luis , Finkelstein Alexei V. , Lockhart Andrew , Merkl Rainer , Perry L. Jeanne , Emery Stephen C. , Gaboriaud Christine , Marks Cara , Moult John , Verlinde Christophe , Eberhard Marc , Elofsson Arne , Hubbard Tim J. P. , Regan Lynne , Banks Jay , Jappelli Roberto , Lesk Arthur M. , Tramontano Anna 
Published: May 2005

AbstractWhat is the current state of the art in protein design? This question was approached in a recent two‐week protein design workshop sponsored by EMBO and held at the EMBL in Heidelberg. The goals were to test available design tools and to explore new design strategies. Five novel proteins were designed: Shpilka, a sandwich of two four‐stranded β‐sheets, a scaffold on which to explore variations in loop topology; Grendel, a four‐helical membrane anchor, ready for fusion to water‐soluble functional domains; Fingerclasp, a dimer of interdigitating β–β–α units, the simplest variant of the “handshake” structural class; Aida, an antibody binding surface intended to be specific for flavodoxin; Leather—a minimal NAD binding domain, extracted from a larger protein. Each design is available as a set of three‐dimensional coordinates, the corresponding amino acid sequence and a set of analytical results. The designs are placed in the public domain for scrutiny, improvement, and possible experimental verification.
Domain flexibility in aspartic proteinases
Authors: Šali Andrej , Veerapandian B. , Cooper Jon B. , Moss David S. , Hofmann Theo , Blundell Tom L. 
Published: May 2005

AbstractComparison of the three‐dimensional structures of native endothiapepsin (EC 3.4.23.6) and 15 endothiapepsin oligopeptide inhibitor complexes defined at high resolution by X‐ray crystallography shows that endothiapepsin exists in two forms differing in the relative orientation of a domain comprising residues 190–302. There are relatively few interactions between the two parts of the enzyme; consequently, they can move as separate rigid bodies. A translational, librational, and screw analysis of the thermal parameters of endothiapepsin also supports and model in which the two parts can move relative to each other. In the comparison of different aspartic proteinases, the rms values are reduced by up to 47% when the two parts of the structure are superposed independently. This justifies description of the differences, including those between pepsinogen and pepsin (EC 3.4.34.1), as a rigid movement of one part relative to another although considerable distortions within the domains also occur. The consequence of the rigid body movement is a change in the shape of the active site cleft that is largest around the S3 pocket. This is associated with a different position and conformation of the inhibitors that are bound to the two endothiapepsin forms. The relevance of these observations to a model of the hydrolysis by aspartic proteinases is briefly discussed.
Deconvolution of the circular dichroism spectra of proteins: The circular dichroism spectra of the antiparallel β‐sheet in proteins
Authors: Perczel A. , Park K. , Fasman Gerald D. 
Published: May 2005

AbstractA recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure β‐pleated sheet in globular proteins. On the basis of X‐ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:‐669–679, 1991), was improved by the addition of proteins with high β‐pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (α‐helix, antiparallel β‐pleated sheet, β‐turns, and unordered conformation), as well as a curve attributed to the “aromatic contribution” in the wavelength range of 195–240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X‐ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X‐ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the α‐helical structure, but upon analysis yielded a considerable amount of β‐sheet in agreement with the X‐ray structure. © 1992 Wiley‐Liss, Inc.
Mutating the charged residues in the binding pocket of cellular retinoic acid‐binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability
Authors: Zhang Jianhua , Liu Zhi‐Ping , Jones T. Alwyn , Gierasch Lila M. , Sambrook Joseph F. 
Published: May 2005

AbstractThree‐dimensional modeling of the complex between retinoic acid‐binding protein (CRABP) and retinoic acid suggests thatbinding of the ligand is mediated by interaction between the carboxyl groupof retinoic acid and two charged amino acids (Arg‐111 and Arg‐131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein–ligand interaction, amino acid substitutions were made by oligonucleotide‐directed, site‐specific mutagenesis. The wild‐type and mutant proteins were expressed in E. coli and subsequently purified. Like wild‐type CRABP, the mutant proteins are composed mainly of β‐strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild‐type protein. Mutants in which Arg‐111 and Arg‐131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild‐type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild‐type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP–retinoic acid complex. These data suggest a model for CRABP–retinoic acid interaction in which the repulsive forces between the positively‐charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively‐charged pair of amino acids produces a protein that is more thermostable than wild‐type CRABP but less effective at ligand‐binding. © 1992 Wiley‐Liss, Inc.
Continuum electrostatics of the C‐peptide: Anatomy of the problem
Authors: Rashin Alexander A. 
Published: May 2005

AbstractA computational study of the role of all ionizable groups of the C‐peptide in its helix–coil transition is performed within the framework of continuum electrostatics. The method employed in our computations involves a numeric solution of the Poisson equation with the Boundary Element Method. Our calculations correctly predict the experimentally observed trends in the helix–coil equilibrium of the C‐peptide, and suggest that the mechanisms involved are more complex than usually presumed in the literature. Our results suggest that electrostatic interactions in the unfolded conformation are often more important than in the helix, total electrostatic contribution to the helix–coil transition due to the side chains of the C‐peptide destabilizes the helix, changes in the helix stability produced by the changes in the ionization state of the side chains are dominated by side chain effects, the effect of the helix dipole on the energetics of the helix–coil transition of the C‐peptide is either minor or similar to other contributions in magnitude; while the formation of a salt bridge is electrostatically favorable, formation of the hydrogen bond between a charged and a polar side chains is not. Factors limiting the accuracy of the computations are discussed. © 1992 Wiley‐Liss, Inc.
Michaelis complexes of porcine pancreatic elastase with 7‐[(alkylcarbamoyl)amino]‐4‐chloro‐3‐ethoxyisocoumarins: Translational sampling of inhibitor position and kinetic measurements
Authors: Plaskon R. Richard , Kam Chih‐Min , Burgess Edward M. , Powers James C. , Suddath F. L. 
Published: May 2005

AbstractA step leading to the formation of the covalent complexes between porcine pancreatic elastase (PPE) and 7‐[(alkylcarbamoyl)amino]‐4‐chloro‐3‐ethoxyisocoumarins (alkylHNCO‐EICs) is the formation of the non‐covalent Michaelis complex. No average structures are available for the Michaelis complexes of PPE with alkylHNCO‐EICs. We present the results of an initial step in obtaining these structures and have determined kinetic constants as well. The kinetic results indicate that formation of the Michaelis complex is what differentiates the effectiveness of these inhibitors in inactivating PPE. The structural and kinetic results together suggest that the structure of the Michaelis complex is necessary for the design of potent alkylHNCO‐EIC inhibitors of PPE. Two novel alkylHNCO‐EICs are predicted to be the best inhibitors of this series. An alternate mechanism for serine protease inhibition is also proposed. Evidence for, and studies that may add support to, the hypothesized mechanism are discussed. © 1992 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus
Authors: Huang Kui , Kodandapani R. , Kallwass Helmut , Hogan James K. , Parris Wendy , Friesen James D. , Gold Marvin , Jones J. Bryan , James Michael N. G. 
Published: May 2005

AbstractBacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three‐dimensional structure solved, the gene coding for it has been cloned, and the proteincan be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4in 50 mM piperazine HCI buffer. The crystals belong to space group P6622 (P6622) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74–92, 1990) crystallized in the space group (P61) with a = 102.3 Å, c = 168.6 Å for the R171W, Q102R, C97G triple mutant, and a = 98.2 Å; c = 162.1 Å for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one‐quarter of a tetramer (Mr 135,000)in the asymmetric unit and have (VM values of 3.8 and 3.3 Å3/dalton, respectively). The R171W mutant diffracts to 2.5 Å and the R171Y mutant to approximately 3.5 Å © 1992 Wiley‐Liss, Inc.