bluuebunny/crossref_metadata_2025 huggingface dataset:
Database of homology‐derived protein structures and the structural meaning of sequence alignment
Authors: Sander Chris , Schneider Reinhard
Published: May 2005

The database of known protein three‐dimensional structures can be significantly increased by the use of sequence homology, based on the following observations. (1) The database of known sequences, currently at more than 12,000 proteins, is two orders of magnitude larger than the database of known structures. (2) The currently most powerful method of predicting protein structures is model building by homology. (3) Structural homology can be inferred from the level of sequence similarity. (4) The threshold of sequence similarity sufficient for structural homology depends strongly on the length of the alignment. Here, we first quantify the relation between sequence similarity, structure similarity, and alignment length by an exhaustive survey of alignments between proteins of known structure and report a homology threshold curve as a function of alignment length. We then produce a database of homology‐derived secondary structure of proteins (HSSP) by aligning to each protein of known structure all sequences deemed homologous on the basis of the threshold curve. For each known protein structure, the derived database contains the aligned sequences, secondary structure, sequence variability, and sequence profile. Tertiary structures of the aligned sequences are implied, but not modeled explicity. The database effectively increases the number of known protein structures by a factor of five to more than 1800. The results may be useful in assessing the structural significance of matches in sequence database searches, in deriving preferences and patterns for structure prediction, in elucidating the structural role of conserved residues, and in modeling three‐dimensional detail by homology.
Role of conserved proline residues in stabilizing tryptophan synthase α subunit: Analysis by mutants with alanine or glycine
Authors: Yutani Katsuhide , Hayashi Seiko , Sugisaki Yoshiko , Ogasahara Kyoko
Published: May 2005

To study the role of Pro residues in the conformation and conformational stability of a protein, nine mutant α subunits of tryptophan synthase from Escherichia coli, in which Ala or Gly was substituted for each of six Pro residues (positions 28, 57, 62, 96, 132, and 207) that are conserved in 10 microoganisms, were constructed by means of site‐directed mutagenesis. The far‐ultraviolet (UV) CD spectra of five mutant α subunits with Ala in place of Pro were identical to the spectrum of the wild‐type protein, the exception being the mutant at position 207 (P207A). CD values in the far‐UV region were less negative for P207A, indicating that the Pro residue at position 207 plays a role in maintaining the intact structure of the α subunit. The negative CD values of the Gly mutants examined (P28G, P96G, and P132G) were also decreased. Calorimetric measurements showed that the two mutants at position 28 (P28G and P28A) gave two peaks in the excess heat capacity curve, whereas the wild type and other Pro mutants had only a single peak. The stability of each mutant protein relative to that of the wild type was about the same for P57A, less for P62A and P132A, and markedly decreased for P96A and P207A, which are substituted at less mobile positions. The changes of denaturation entropy (ΔΔdS) at the denaturation temperature of the wild‐type protein (54.1 °C at pH 9.0) were positive for P57A, P62A, and P132A, but negative for P96A, P207A, and P132G. The present results do not indicate that the differences in stability (ΔdG) among Pro substitutions are caused only by an entropic factor, as might be theoretically expected. The decreases in stability for P96A and P207 were due to the considerable decrease in denaturation enthalpy, although they were partly compensated for by the decrease in entropy. Our results also suggest that Pro‐28 stabilizes the interaction between two domains of the α subunit.
The isolation, purification, and preliminary crystallographic characterization of udp‐galactose‐4‐epimerase from Escherichia coli
Authors: Bauer Alan J. , Rayment Ivan , Frey Perry A. , Holden Hazel M.
Published: May 2005

Uridine diphosphogalactose‐4‐epimerase from E. coli has been crystallized in a form suitable for a high‐resolution X‐ray crystallographic structural analysis. The enzyme complexed with a substrate analogue, uridine diphosphobenzene (UDP‐benzene), crystallizes readily using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions, a = 76.3 Å, b = 83.1 Å, and c = 132.1 Å. Based on still setting photographs, the crystals diffract to a nominal resolution of 2.3 Å and are stable in the X‐ray beam. The enzyme used in these experiments was produced by a new expression system and a modified purification scheme.
Preliminary x‐ray analysis of crystals of plasminogen activator inhibitor‐1
Authors: Goldsmith Elizabeth J. , Sheng‐Cheng Chen , Danley Dennis E. , Gerard Robert D. , Geoghegan Kieran F. , Mottonen James , Strand Arne
Published: May 2005

Crystals of bacterially expressed plasminogen activator inhibitor (PAI‐1) suitable for X‐ray diffraction analysis have been obtained from 8% (w/v) PEG 1500, pH 8.25. The space group is P1, and the lattice constants are a = 82.17 Å, b = 47.82 Å, c = 62.89 Å, α = 90.00°, β = 106.90°, γ = 106.84°. The diffraction limit is 2.3 Å, and the unit cell contains two molecules of PAI‐1. The crystals contain latent PAI‐1 which can be partly reactivated by exposure to denaturants.
Hydrophobic clustering in nonnative states of a protein: Interpretation of chemical shifts in NMR spectra of denatured states of lysozyme
Authors: Evans Philip A. , Topping Karen D. , Woolfson Derek N. , Dobson Christopher M.
Published: May 2005

Chemical shifts of resonances of specific protons in the 1H NMR spectrum of thermally denatured hen lysozyme have been determined by exchange correlation with assigned native state resonances in 2D NOESY spectra obtained under conditions where the two states are interconverting. There are subtle but widespread deviations of the measured shifts from the values which would be anticipated for a random coil; in the case of side chain protons these are virtually all net upfield shifts and it is shown that this may be the averaged effect of interactions with aromatic rings in a partially collapsed denatured state. In a very few cases, notably that of two sequential tryptophan residues, it is possible to interpret these effects in terms of specific, local interresidue interactions. Generally, however, there is no correlation with either native state shift perturbations or with sequence proximity to aromatic groups. Diminution of most of the residual shift perturbations on reduction of the disulfide cross‐links confirms that they are not simply effects of residues adjacent in the sequence. Similar effects of chemical denaturants, with the disulfides intact, demonstrate that the shift perturbations reflect an enhanced tendency to side chain clustering in the thermally denatured state. The temperature dependences of the shift perturbations suggest that this clustering is noncooperative and is driven by small, favorable enthalpy changes. While the extent of conformational averaging is clearly much greater than that observed for a homologous protein, α‐lactalbumin, in its partially folded “molten globule” state, the results clearly show that thermally denatured lysozyme differs substantially from a random coil, principally in that it is partially hydrophobically collapsed.
The crystal structure of staphylococcal nuclease refined at 1.7 Å resolution
Authors: Hynes Thomas R. , Fox Robert O.
Published: May 2005

The crystal structure of staphylococcal nuclease has been determined to 1.7 Å resolution with a final R‐factor of 16.2% using stereochemically restrained Hendrickson–Konnert least‐squares refinement. The structure reveals a number of conformational changes relative to the structure of the ternary complex of staphylococcal nuclease1,2 bound with deoxythymidine‐3′,5′‐diphosphate and Ca2+. Tyr‐113 and Tyr‐115, which pack against the nucleotide base in the nuclease complex, are rotated outward creating a more open binding pocket in the absence of nucleotide. The side chains of Ca2+ ligands Asp‐21 and Asp‐40 shift as does Glu‐43, the proposed general base in the hydrolysis of the 5′‐phosphodiester bond. The significance of some changes in the catalytic site is uncertain due to the intrusion of a symmetry related Lys‐70 side chain which hydrogen bonds to both Asp‐21 and Glu‐43. The position of a flexible loop centered around residue 50 is altered, most likely due to conformational changes propagated from the Ca2+ site. The side chains of Arg‐35, Lys‐84, Tyr‐85, and Arg‐87, which hydrogen bond to the 3′‐ and 5′‐phosphates of the nucleotide in the nuclease complex, are unchanged in conformation, with packing interactions with adjacent protein side chains sufficient to fix the geometry in the absence of ligand. The nuclease structure presented here, in combination with the stereochemically restrained refinement of the nuclease complex structure2 at 1.65 Å, provides a wealth of structural information for the increasing number of studies using staphylococal nuclease as a model system of protein structure and function.
1.59 Å structure of trypsin at 120 K: Comparison of low temperature and room temperature structures
Authors: Earnest Thomas , Fauman Eric , Craik Charles S. , Stroud Robert
Published: May 2005

The structure of a rat trypsin mutant [S195C] at a temperature of 120 K has been refined to a crystallographic R factor of 17.4% between 12.0 and 1.59 Å and is compared with the structure of the D102N mutant at 295 K. A reduction in the unit cell dimensions in going from room temperature to low temperature is accompanied by a decrease in molecular surface area and radius of gyration. The overall structure remains similar to that at room temperature. The attainable resolution appears to be improved due to the decrease in the fall off of intensities with resolution [reduction of the temperature factor]. This decreases the uncertainty in the atomic positions and allows the localization of more protein atoms and solvent molecules in the low temperature map. The largest differences between the two models occur at residues with higher than average temperature factors. Several features can be localized in the solvent region of the 120 K map that are not seen in the 295 K map. These include several more water molecules as well as an interstitial sulfate ion and two interstitial benzamidine molecules.
On the multiple‐minima problem in the conformational analysis of polypeptides. V. Application of the self‐consistent electrostatic field and the electrostatically driven monte carlo methods to bovine pancreatic trypsin inhibitor
Authors: Ripoll Daniel R. , Piela Lucjan , Váasquez Max , Scheraga Harold A.
Published: May 2005

In connection with the accompanying paper to test various models for the hydration of polypeptides, we have explored a limited portion of the conformational energy hyperspace of the small protein bovine pancreatic trypsin inhibitor (BPTI) with the aid of two search methods developed in this laboratory. A series of low‐energy conformations was obtained as a result of this study. These conformations constitute a set of local minima in the conformational energy space of the molecule as described by the ECEPP/2 (Empirical Conformational Energy Program for Peptides) potential energy function, without the inclusion of hydration. Five different initial conformations were used in this exploration: the first corresponds to an energy‐refined structure based on the crystallographic coordinates (4PTI) provided by Deisenhofer and Steigemann25 and reported previously by Meirovitch and Scheraga.3 The remaining four initial conformations were obtained by using a Variable‐Target‐Function procedure, applied to the experimental Cartesian coordinates (5PTI) reported by Wlodawer et al.26

The self‐consistent electrostatic field (SCEF) and the electrostatically driven Monte Carlo (EDMC) methods were used to search the conformational space. The SCEF and EDMC methodologies assume that a polypeptide or protein molecule is driven toward the native structure mainly by the action of the electrostatic interactions. Application of these methodologies led to a set of conformations (up to 50 kcal/mol lower than the starting ones) with ECEPP/2 energies lower than any of those that we had previously found. Application of both methods to the initial conformation generated from 4PTI led to a series of low‐energy conformations exhibiting similar rms deviations with respect to the experimental data (4PTI) as did the starting conformation. However, statistical analysis of the runs that had started from the conformations generated by using the variable‐target‐function procedure (and applying the EDMC method) indicated that the rms deviations of the atomic positions of the new low‐energy conformations tended to increase as the energy improved, when compared with the X‐ray data from which the starting conformations had been generated. The structures with the lowest energies also had radii of gyration smaller than the experimentally observed one. These results indicated a need to include hydration in the potential function, and provided the conformations used in the accompanying paper to test various hydration models.
Empirical solvation models can be used to differentiate native from near‐native conformations of bovine pancreatic trypsin inhibitor
Authors: Vila J. , Williams R. L. , Vásquez M. , Scheraga H. A.
Published: May 2005

Several hydration models for peptides and proteins based on solvent accessible surface area have been proposed previously. We have evaluated some of these models as well as four new ones in the context of near‐native conformations of a protein. In addition, we propose an empirical site–site distance‐dependent correction that can be used in conjuction with any of these models.

The set of near‐native structures consisted of 39 conformations of bovine pancreatic trypsin inhibitor (BPTI) each of which was a local minimum of an empirical energy function (ECEPP) in the absence of solvent. Root‐mean‐square (rms) deviations from the crystallographically determined structure were in the following ranges: 1.06–1.94 Å for all heavy atoms, 0.77–1.36 Å for all backbone heavy atoms, 0.68–1.33 Å for all α‐carbon atoms, and 1.41–2.72 Å for all side‐chain heavy atoms.

We have found that there is considerable variation among the solvent models when evaluated in terms of concordance between the solvation free energy and the rms deviations from the crystallographically determined conformation. The solvation model for which the best concordance (0.939) with the rms deviations of the Cα atoms was found was derived from NMR coupling constants of peptides in water combined with an exponential site–site distance dependence of the potential of mean force.

Our results indicate that solvation free energy parameters derived from nonpeptide free energies of hydration may not be transferrable to peptides. Parameters derived from peptide and protein data may be more applicable to conformational analysis of proteins. A general approach to derive parameters for free energy of hydration from ensemble‐averaged properties of peptides in solution is described.
Proline in α‐helix: Stability and conformation studied by dynamics simulation
Authors: Yun R. H. , Anderson Amil , Hermans Jan
Published: May 2005

Free‐energy simulations have been used to estimate the change in the conformational stability of short polyalanine α‐helices when one of the alanines is replaced by a proline residue. For substituting proline in the middle of the helix the change in free energy of folding (ΔΔG°) was calculated as 14 kJ/mol (3.4 kcal/mol), in excellent agreement with the one available experimental value. The helix containing proline was found to be strongly kinked; the free energy for reducing the angle of the kink from 40° to 15° was calculated, and found to be small. A tendency to alternate hydrogen bonding schemes was observed in the proline‐containing helix. These observations for the oligopeptide agree well with the observation of a range of kink angles (18–35°) and variety of hydrogen bonding schemes, in the rare instances where proline occurs in helices in globular proteins. For substituting proline at the N‐terminus of the helix the change in free energy of folding (ΔΔG°) was calculated as −4 kJ/mol in the first helical position (N1) and +6 kJ/mol in the second helical position (N2). The observed frequent occurrence of proline in position N1 in α‐helices in proteins therefore has its origin in stability differences of secondary structure. The conclusion reached here that proline may be a better helix former in position N1 than (even) alanine, and thus be a helix initiator may be testable experimentally by measurements of fraction helical conformation of individual residues in oligopeptides of appropriate sequence. The relevance of these results in regards to the frequent occurrence of proline‐containing helices in certain membrane proteins is discussed.
Structure of ricin A‐chain at 2.5 Å
Authors: Katzin Betsy J. , Collins Edward J. , Robertus Jon D.
Published: May 2005

Ricin has been refined in a crystallographic sense to 2.5 Å resolution and the model for the A‐chain (RTA) is described in detail. Because RTA is the first member of the class of plant toxins to be analyzed, this model probably defines the major structural characteristics of the entire family of these medically important proteins. Explanations are provided to rationalize amino acids that are conserved between RTA and a number of homologous plant and bacterial toxins. Eight invariant residues appear to be involved in creating or stabilizing the active site. In the active site Arg180 and Glu177 are hydrogen bonded to each other and also coordinate a water molecule; each of these groups may be important in the N‐glycosidation reaction. Several other polar residues may play lesser roles in the mechanism, including tyrosines 80 and 123 and asparagines 78 and 209. A number of conserved hydrophobic residues are seen to cluster within several patches and probably drive the overall folding of the toxin molecule.
Site‐directed mutagenesis of ricin A‐chain and implications for the mechanism of action
Authors: Ready Michael P. , Kim Youngsoo , Robertus Jon D.
Published: May 2005

Ricin A‐chain is an N‐glycosidase that attacks ribosomal RNA at a highly conserved adenine residue. The enzyme is representative of a large family of medically significant proteins used in the design of anticancer agents and in the treatment of HIV infection. The x‐ray structure has been used as a guide to create several active site mutations by directed mutagenesis of the cloned gene. Glu177 is a key catalytic residue, and conversion to Gln reduces activity 180‐fold. Asn209 is shown to participate in substrate binding by kinetic analysis. Conversion to Ser increases Km sixfold but has no effect on kcat. Conversion of Tyr80 and Tyr123 to Phe decreases activity by 15‐ and 7‐fold respectively. A mechanism of action is proposed that involves binding of the substrate adenine in a syn configuration that resembles the transition state; the putative oxycarbonium ion is probably stabilized by interaction with Glu177.
Physical reasons for secondary structure stability: α‐Helices in short peptides
Authors: Finkelstein A. V. , Badretdinov A. Y. , Ptitsyn O. B.
Published: May 2005

It was recently found that some short peptides (including C‐ and S‐peptide fragments of RNase A) can have considerable helicity in solution, 1–12 which was considered to be surprising. Does the observed helicity require a new explanation, or is it consistent with previous understanding? In this work we show that this helicity is consistent with the physical theory of secondary structure12–19 based on an extension of the conventional Zimm‐Bragg model.20 Without any special modifications, this theory explains reasonably well almost all the experimentally observed dependencies of helicity on pH, temperature, and amino acid replacements. We conclude that the observed “general level” of helicity of C‐ and S‐peptides (5–30% at room temperature and 10–50% near 0°C) is “normal” for short peptides consisting mainly of helix‐forming and helix‐indifferent residues. The helicity is modified by a multitude of weak specific side chain interactions, many of which are taken into account by the present theory;13–19 some discrepancies between the theory and experiment can be explained by weak side‐chain‐side chain interactions that were neglected. A reasonable coincidence of the theory with experiment suggests that it had been used to investigate the role of local interactions in the formation of α‐helical “embryos” in unfolded protein chains.
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

The 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

A 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

Calcium 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

Crystal 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

The 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

Nonglycosylated 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

Quantum‐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

We 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

A 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

Pig 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

Diffuse 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

The 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

While 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

In 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

The 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

In 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.
Amino acid substitution analysis of E. coli thymidylate synthase: The study of a highly conserved region at the N‐terminus
Authors: Kim Choll Wan , Michaels Mark Leo , Miller Jeffrey H.
Published: May 2005

Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure‐function relationship of the Escherichia coli TS protein at the N‐terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a β‐strand. We find that residues surrounding a β‐bulge structure within the β‐strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested. © 1992 Wiley‐Liss, Inc.
Structural effects induced by mutagenesis affected by crystal packing factors: The structure of a 30–51 disulfide mutant of basic pancreatic trypsin inhibitor
Authors: Eigenbrot Charles , Randal Michael , Kossiakoff Anthony A.
Published: May 2005

The X‐ray structure of the C30V/C51A disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) has been analyzed at 1.6 Å resolution. The mutant crystallizes in a cell having two molecules in the asymmetric unit. The packing environments of these two molecules are quite different, allowing for an assessment of which among the observed structural changes result from the mutation and which are produced by lattice packing considerations. The removal of the 30–51 disulfide bridge has little apparent affect on the B‐factors of segments of adjacent polypeptide chain, although there are distinct differences in the structure compared to wild‐type BPTI crystal structures. Both of the two C30V/C51A molecules show differences at the mutation site when compared to another 30–51 disulfide mutant, C30A/C51A, presumably due to the larger steric bulk of a valine versus an alanine at residue 30. A comparison of the two independent C30V/C51A molecules indicates that there are significant differences between them even at the site of mutation. The description of the specific structural differences of each molecule differs in detail and suggests different conclusions about the nature of structural perturbation near 30–51. In addition, when these two molecules are compared to two different wild‐type structures, which had been determined from different space groups, a somewhat different pattern of changes is observed. These findings indicate that crystal packing can influence the observed perturbations in mutant structures. © 1992 Wiley‐Liss, Inc.