bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conformational changes in yeast phosphoglycerate kinase upon ligand binding: Fluorescence of a linked probe and chemical reactivity of genetically introduced cysteinyl residues
Authors: Desmadril Michel , Minard Philippe , Ballery Nathalie , Gaillard‐Miran Sophie , Hall Len , Yon Jeannine M. 
Published: May 2005

AbstractThe effects of ligands on the conformation of yeast phosphoglycerate kinase were explored by introducing cysteinyl residues at different positions in the molecule by site‐directed mutagenesis. Thus several mutants were constructed, each containing a unique cysteinyl residue. Neither the conformation nor the enzyme activity was affected by the substitutions. The reactivity of the thiol groups and the fluorescence of N‐acetyl‐N′‐(5‐sulfo‐1‐naphtyl)ethylene‐diamine covalently linked to these thiols were used to monitor the conformational changes induced upon ligand binding.It was found that the observed changes mainly involve the part of the protein located in the cleft, particularly the environment of residues 35 and 183. No alteration was observed on the external side of the protein. Only 3‐Phosphoglycerate induced these conformational changes. However, when the fluorescent probe was attached to residue 377, the binding of the two substrates was required to induce a modification in the fluorescence of the probe. These results indicate that the substrates separately or together induce discrete molecular motions in phosphoglycerate kinase.
Functionality maps of binding sites: A multiple copy simultaneous search method
Authors: Miranker Andrew , Karplus Martin 
Published: May 2005

AbstractA new method is proposed for determining energetically favorable positions and orientations for functional groups on the surface of proteins with known three‐dimensional structure. From 1,000 to 5,000 copies of a functional group are randomly placed in the site and subjected to simultaneous energy minimization and/or quenched molecular dynamics. The resulting functionality maps of a protein receptor site, which can take account of its flexibility, can be used for the analysis of protein ligand interactions and rational drug design. Application of the method to the sialic acid binding site of the influenza coat protein, hemagglutinin, yields functional group minima that correspond with those of the ligand in a cocrystal structure.
A model for human cardiac troponin C and for modulation of its Ca2+ affinity by drugs
Authors: Ovaska Martti , Taskinen Jyrki 
Published: May 2005

AbstractCalcium sensitizers are drugs which increase force development in striated muscle by sensitizing myofilaments to Ca2+. This can happen by increasing Ca2+ affinity of the regulatory domain of Ca2+ binding protein troponin C. High resolution crystal structures of two calcium binding proteins, calmodulin (Babu et al.: J. Mol. Biol. 203:191–204, 1988) and skeletal troponin C (Satyshur et al.: J. Biol. Chem. 263:1628–1647, 1988; Herzber et al.: J. Mol. Biol. 203:761–779, 1988), have recently been published. This makes it possible to model in detail the calcium‐sensitizing action of drugs on troponin C.In this study a model of human cardiac troponin C in three‐calcium state has been constructed. When calcium is bound to calcium site II of cardiac troponin C an open conformation of the protein results, which has a hydrophobic pocket surrounded by a few polar side chains. Complexation of three drugs, trifluoperazine, bepridil, and pimobendan, to the hydrophobic pocket is studied using energy minimization techniques. Two different binding modes are found, which differ in the location of a strong electrostatic interaction. In analogy with the crystal structure of skeletal troponin C it is hypothezed that in cardiac troponin C an interaction occurs between Gln‐50 and Asp‐88, which has a long‐range effect on calcium binding. The binding modes of drugs, where a strong interaction with Asp‐88 exists, can effectively prevent the interaction between Asp‐88 and Gln‐50 in the protein, and are proposed to be responsible for the calcium‐sensitizing properties of the studied drugs.
An autoantibody to single‐stranded DNA: Comparison of the three‐dimensional structures of the unliganded fab and a deoxynucleotide–fab complex
Authors: Herron J. N. , He X. M. , Ballard D. W. , Blier P. R. , Pace P. E. , Bothwell A. L. M. , Voss E. W. , Edmundson A. B. 
Published: May 2005

AbstractCrystal structures of the Fabs from an autoantibody (BV04‐01) with specificity for single‐stranded DNA have been determined in the presence and absence of a trinucleotide of deoxythymidylic acid, d(pT)3. Formation of the ligand‐protein complex was accompanied by small adjustments in the orientations of the variable (VL and VH) domains. In addition, there were local conformational changes in the first hypervariable loop of the light chain and the third hypervariable loop of the heavy chain, which together with the domain shifts led to an improvement in the complementarity of nucleotide and Fab. The sugar–phosphate chain adopted an extended and “open” conformation, with the base, sugar, and phosphate components available for interactions with the protein. Nucleotide 1 (5′‐end) was associated exclusively with the heavy chain, nucleotide 2 was shared by both heavy and light chains, and nucleotide 3 was bound by the light chain. The orientation of phosphate 1 was stabilized by hydrogen bonds with serine H52a and asparagine H53. Phosphate 2 formed an ion pair with arginine H52, but no other charge–charge interactions were observed. Insertion of the side chain of histidine L27d between nucleotides 2 and 3 resulted in a bend in the sugar–phosphate chain. The most dominant contacts with the protein involved the central thymine base, which was immobilized by cooperative stacking and hydrogen bonding interactions. This base was intercalated between a tryptophan ring (no. H100a) from the heavy chain and a tyrosine ring (no. L32) from the light chain. The resulting orientation of thymine was favorable for the simultaneous formation of two hydrogen bonds with the backbone carbonyl oxygen and the side chain hydroxyl group of serine L91 (the thymine atoms were the hydrogen on nitrogen 3 and keto oxygen 4).
Analysis of the steric strain in the polypeptide backbone of protein molecules
Authors: Herzberg Osnat , Moult John 
Published: May 2005

AbstractThe extent to which local strain is present in the polypeptide backbone of folded protein molecules has been examined. The occurrence of steric strain associated with nonproline cis peptide bonds and energetically unfavorable main chain dihedral angles can be identified reliably from the well ordered parts of high resolution, refined crystal structures. The analysis reveals that there are relatively few sterically strained features. Those that do occur are located overwhelmingly in regions concerned with function. We attribute this to the greater precision necessary for ligand binding and catalysis, compared with the requirements of satisfactory folding.
Crystallization and preliminary X‐ray diffraction studies of human salivary α‐amylase
Authors: Ramasubbu Narayanan , Bhandary Krishna K. , Scannapieco Frank A. , Levine Michael J. 
Published: May 2005

AbstractNonglycosylated α‐amylase, a major component of human parotid saliva, has been crystallized by the vapor diffusion technique using 2‐methyl‐2,4‐pentanediol as the precipitant in the presence of CaCl2 at pH 9.0. The crystals are orthorhombic, space group P212121 with unit cell dimensions of a = 53.3, b = 75.8, and c = 138.1 Å. The asymmetric unit contains one amylase molecule. The solvent content is 54%. The crystals are stable to X‐rays and diffract up to 2.8 Å and appear to be suitable for X‐ray diffraction studies.
Changes in the electron density of the cofactor NADPH on binding to E. coli dihydrofolate reductase
Authors: Bajorath Jürgen , Li Zhenqin , Fitzgerald George , Kitson David H. , Farnum Martin , Fine Richard M. , Kraut Joseph , Hagler Arnold T. 
Published: May 2005

AbstractQuantum‐mechanical electron density calculations reveal that a significant polarization is induced in the cofactor NADPH (reduced nicotinamide adenine dinucleotide phosphate) on binding to the enzyme dihydrofolate reductase. The calculations indicate that electron density corresponding to ∼0.7 electron charges is shifted within the molecule, extending over more than 20Å. Further calculations on proposed enzyme mutants show that the polarization of NADPH on binding to DHFR is, in large part, induced by a motif of three positively charged residues. This motif was also identified to be directly responsible for the positive electrostatic potential surrounding the cofactor binding site in the enzyme. The possibility of this long‐range polarization of NADPH was originally proposed based on a previous study of ligand binding to DHFR where a conserved structural motif of three positively charged residues was found to play a major role in polarizing the substrate folate over its entire length of 18 Å.
Conformational analysis of a 12‐residue analogue of mastoparan and of mastoparan X
Authors: Faerman Carlos H. , Ripoll Daniel R. 
Published: May 2005

AbstractWe have investigated the conformational properties of a truncated analogue of mastoparan and of mastoparan X, both peptides from wasp venom. The electrostatically driven Monte Carlo method was used to explore the conformational space of these short peptides. The initial conformations used in this study, mainly random ones, led to α‐helical conformations. The α‐helical conformations thus found exhibit an amphipathic character. These results are in accord with experimental data from NMR and CD spectroscopy.
Electrostatic fields at the active site of ribulose‐1,5‐bisphosphate carboxylase
Authors: Lu Guoguang , Lindqvist Ylva , Schneider Gunter 
Published: May 2005

AbstractA macroscopic approach has been employed to calculate the electrostatic potential field of nonactivated ribulose‐1,5‐bis‐phosphate carboxylase and of some complexes of the enzyme with activator and substrate. The overall electrostatic field of the L2‐type enzyme from the photosynthetic bacterium Rhodospirillum rubrum shows that the core of the dimer, consisting of the two C‐terminal domains, has a predominantly positive potential. These domains provide the binding sites for the negatively charged phosphate groups of the substrate. The two N‐terminal domains have mainly negative potential. At the active site situated between the C‐terminal domain of one subunit and the N‐terminal domain of the second subunit, a large potential gradient at the substrate binding site is found. This might be important for polarization of chemical bonds of the substrate and the movement of protons during catalysis. The immediate surroundings of the activator lysine, K191, provide a positive potential area which might cause the pK value for this residue to be lowered. This observation suggests that the electrostatic field at the active site is responsible for the specific carbamylation of the ε‐amino group of this lysine side chain during activation. Activation causes a shift in the electrostatic potential at the position of K166 to more positive values, which is reflected in the unusually low pK of K166 in the activated enzyme species. The overall shape of the electrostatic potential field in the L2 building block of the L8S8‐type Rubisco from spinach is, despite only 30% amino acid homology for the L‐chains, strikingly similar to that of the L2‐type Rubisco from Rhodospirillum rubrum. A significant difference between the two species is that the potential is in general more positive in the higher plant Rubisco. In particular, the second phosphate binding site has a considerably more positive potential, which might be responsible for the higher affinity for the substrate of L8S8‐type enzymes. The higher potential at this site might be due to two remote histidine residues, which are conserved in the plant enzymes.
Crystal structure of the binary complex of pig muscle phosphoglycerate kinase and its substrate 3‐phospho‐D‐glycerate
Authors: Harlos Karl , Vas Maria , Blake Colin F. 
Published: May 2005

AbstractPig muscle phosphoglycerate kinase has been crystallized from polyethyleneglycol in the presence of its substrate 3‐phospho‐D‐glycerate (3‐PG) and the structure has been determined at 2.0 Å resolution. The structure was solved using the known structure of the substrate‐free horse muscle enzyme and has been refined to a crystallographic R‐factor of 21.5%. 3‐Phospho‐D‐glycerate is bound to the N‐domain of the enzyme through a network of hydrogen bonds to a cluster of basic amino acid residues and by electrostatic interactions between the negatively charged phosphate and these basic protein side chains. This binding site is in good agreement with earlier proposals [Banks et al., Nature (London) 279:773–777, 1979]. The phosphate oxygen atoms are hydrogen bonded to His‐62, Arg‐65, Arg‐122, and Arg‐170. The 2‐hydroxyl group, which defines the D‐isomer of 3PG, is hydrogen bonded to Asp‐23 and Asn‐25. The carboxyl group of 3‐PG points away from the N‐domain towards the C‐domain and is hydrogen bonded via a water molecule to main chain nitrogen atoms of helix‐14. The present structure of the 3‐PG‐bound pig muscle enzyme is compared with the structure of the substrate‐free horse enzyme. Major changes include an ordering of helix‐13 and a domain movement, which brings the N‐domain closer to the ATP‐binding C‐domain. This domain movement consists of a 7.7° rotation, which is less than previously estimated for the ternary complex. Local changes close to the 3‐PG binding site include an ordering of Arg‐65 and a shift of helix‐5.
Correlations of atomic movements in lysozyme crystals
Authors: Clarage James B. , Clarage Michael S. , Phillips Walter C. , Sweet Robert M. , Caspar Donald L. D. 
Published: May 2005

AbstractDiffuse scattering data have been collected on two crystal forms of lysozyme, tetragonal and triclinic, using synchrotron radiation. The observed diffraction patterns were simulated using an exact theory for simple model crystals which relates the diffuse scattering intensity distribution to the amplitudes and correlations of atomic movements. Although the mean square displacements in the tetragonal form are twice that in the triclinic crystal, the predominent component of atomic movement in both crystals is accounted for by short‐range coupled motions where displacement correlations decay exponentially as a function of atomic separation, with a relaxation distance of ≈ 6 Å. Lattice coupled movements with a correlation distance ≈ 50 Å account for only about 5–10% of the total atomic mean square displacements in the protein crystals. The results contradict various presumptions that the disorder in protein crystals can be modeled predominantly by elastic vibrations or rigid body movements.
Integrity of refolded and reoxidized gelatin‐binding fragments of fibronectin
Authors: Ingham K. C. , Brew S. A. 
Published: May 2005

AbstractThe gelatin‐binding region of fibronectin is easily isolated as a stable and functional 42‐kDa fragment (42‐kDa GBF) containing four type I “finger” modules and two type II “kringle‐like” modules arranged in the order I6–II1–II2–I7–I8–I9, where the numbers designate the order of these modules in each of the two polypeptide chains. Each module forms an independently folded domain stabilized by two disulfide bonds. Reduction of disulfides caused large changes in the intrinsic fluorescence and abolished the gelatin‐binding activity of 42‐kDa GBF and two nonoverlapping gelatin‐binding subfragments, 30‐kDa GBF (I6–II1–II2–I7) and 21‐kDa GBF (I8–I9). However, high yields of active material could be regenerated, without diluting the protein, by dialysis into GdmCl followed by slow overnight removal of GdmCl while maintaining the redox potential with a mixture of oxidized and reduced glutathione. Fluorescence spectroscopic analysis indicated that the tertiary structure and thermodynamic stability of the refolded fragments were similar to those of the originals. The refolded fragments were quantitatively indistinguishable from the originals with respect to their dissociation constants for binding to a fluorescent‐labeled collagen fragment. The results suggest that all or most of the cystines, a total of 24 in 42‐kDa GBF, are correctly paired in the refolded products and that the tertiary structure was completely recovered. The fact that the 30‐ and 21‐kDa fragments bind with a similar affinity proves the existence of at least two nonoverlapping sites in 42‐kDa GBF that recognize gelatin.
Comparative analysis of the β transducin family with identification of several new members including PWP1, a nonessential gene of Saccharomyces cerevisiae that is divergently transcribed from NMT1
Authors: Duronio Robert J. , Gordon Jeffrey I. , Boguski Mark S. 
Published: May 2005

AbstractWhile investigating the expression of the Saccharomyces cerevisiae myristoyl‐CoA:protein N‐myristoyltransferase gene (NMT: E.C. 2.3.1.97) by Northern blot analysis, we observed another RNA transcript whose expression resembled that of NMT1 during meiosis and was derived from a gene located <1 kb immediately upstream of NMT1. This new gene, designated PWP1 (for periodic tryptophan protein), is divergently transcribed from NMT1 and encodes a 576‐residue protein. Null mutants of PWP1 are viable, but their‐growth is severely retarded and steady‐state levels of several cellular proteins (including at least two proteins that label with exogenous [3H]myristic acid) are drastically reduced. New methods for database searching and assessing the statistical significance of sequence similarities identic PWP1 as a member of the β‐transducin protein superfamily. Two other previously unrecognized β‐transducin‐like proteins (S. cerevisiae MAKI1 and D. discoideum AAC3) were also identified, and an unexpectedly high degree of sequence homology was found between a Chlamydomonas β‐like polypeptide and the C12.3 gene of chickens. A systematic and quantitative comparative analysis resulted in classifying all β‐transducin‐like sequences into II nonorthologous families. Based on specific sequence attributes, however, not all β‐transducin‐like sequences are expected to be functionally similar, and quantitative criteria for inferring functional analogies are discussed. Possible roles of repetitive tryptophan residues in proteins are also considered. Published 1992 Wiley‐Liss, Inc.
Structural and energetic differences between insertions and substitutions in staphylococcal nuclease
Authors: Sondek John , Shortle David 
Published: May 2005

AbstractIn a previous study, the small protein staphylococcal nuclease was shown to readily accommodate single alanine and glycine insertions, with average losses in stability comparable to substitutions at the same sites (PROT. 7:29–305, 1990). To more fully explore this unexpected adaptability to changes in residue spacing, 2 double amino acid insertions (alanyl‐glycine, glycyl‐glycine) and 3 additional single amino acid insertions with dissimilar side chains (proline, leucine, and glutamine) were constructed at 10 of the sites previously studied. At 8 of these sites, the type of amino acid side chain on the inserted residue significantly influenced the stability of the mutant protein. However, at 9 of the 10 sites, the double insertions were found to be no more destabilizing than the single alanine or glycine insertions. In contrast, double substitution mutations of staphylococcal nuclease, which replace two adjacent residues with alanine, do not show this striking degree of non‐additivity. A comparison of the effects of single glutamine and single glycine insertions with alanyl‐glycine insertions indicates that insertion of alanine into the peptide backbone is, on average, less destabilizing than appending the equivalent atoms onto the side chain of a glycine insertion. To explain their very different energetic effects, we propose that, unlike most substitutions, the inserted residue(s) must induce lateral displacements of the polypeptide chain, forcing the folded conformation away from that of wild type. The resulting obligatory shifts in the positioning of residues flanking the insertion generate a large number of degrees of freedom around which the mutant structure can relax. From the many alternative packing and bonding arrangements thus made available to the polypeptide chain, the energetically most favorable is selected. © 1992 Wiley‐Liss, Inc.
Structures of complexes of rhizopuspepsin with pepstatin and other statine‐containing inhibitors
Authors: Suguna Kaza , Padlan Eduardo A. , Bott Richard , Boger Joshua , Parris Kevin D. , Davies David R. 
Published: May 2005

AbstractThe three‐dimensional structures of the complexes of the aspartic proteinase from Rhizopus chinensis (Rhizopuspepsin, EC 3.4.23.6) with pepstatin and two pepstatin like peptide inhibitors of renin have been detrmined by X‐ray diffraction methods and refined by restrained least‐squares procedures. The inhibitors adopt an extended conformation and lie in the deep groove located between the two domains of the enzyme. Inhibitor binding is accompanied by a conformational change at the “flap,” a β‐hairpin loop regions, that projects over the binding cleft andcloses down over the inhibitor, excluding water molecules from the vicinityof the scissile bond. The hydroxyl group of the central statyl residue of the inhibitors replaces the water molecule found between the two active aspartates, Asp‐35 and Asp‐218, in the native structure. The refined structures provide additional data to define the specific subsites of the enzyme and also show a system of hydrogen bonding to the inhibitor backbone similar to that observed for a reduced inhibitor. Published 1992 Wiley‐Liss, Inc.
Molecular modeling of the amphipathic helices of the plasma apolipoproteins
Authors: Brasseur Robert , Lins Laurence , Vanloo Berlinda , Ruysschaert Jean‐Marie , Rosseneu Maryvonne 
Published: May 2005

AbstractIn this paper we propose a classification of the amphipathic helicalrepeats occurring in the plasma apolipoprotein sequences. It is based upon the calculation of the molecular hydrophobicity potential around the helical segments. The repeats were identified using a new autocorrelation matrix, based upon similarities of hydrophobic and hydrophilic properties of the amino acid residues within the apolipoprotein sequences. The helices were constructed by molecular modeling, the molecular hydrophobicity potential was calculated, and isopotential contour lines drawn around the helices yieldeda three‐dimensional visualization of the hydrophobicity potential. Two classes of apolipoproteins could be differentiated by comparing the hydrophobic angles obtained by projection of the isopotential contour lines on a plane perpendicular to the long axis of the helix. The isopotential contour lines around apo AI, AIV, and E are more hydrophilic than hydrophobic, whereas they are of similar intensity for apo AII, CI, and CIII. In both cases discoidal lipid–protein complexes are generated, with the amphipathic helices around the edge of the lipid core. The long axis of the helices is oriented parallel to the phospholipid acyl chains and the hydrophilic side of the helix toward the aqueous phase. As a result of the differences in hydrophobicity potential, the contact between the hydrophobic side of the helices and the phospholipid acyl chains is larger for apo AII, CI, and CIII than for the other apolipoproteins. This might account for the greater stability of the discoidal complexes generated between phospholipids and these apoproteins. © 1992 Wiley‐Liss, Inc.