bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and functional relations among thioredoxins of different species
Authors: Eklund Hans , Gleason Florence K. , Holmgren Arne
Published: May 2005

Three‐dimensional models have been constructed of homologous thioredoxins and protein disulfide isomerases based on the high resolution x‐ray crystallographic structure of the oxidized form of Escherichia coli thioredoxin. The thioredoxins, from archebacteria to humans, have 27–69% sequence identity to E. coli thioredoxin. The models indicate that all the proteins have similar three‐dimensional structures despite the large variation in amino acid sequences. As expected, residues in the active site region of thioredoxins are highly conserved. These include Asp‐26, Ala‐29, Trp‐31, Cys‐32, Gly‐33, Pro‐34, Cys‐35, Asp‐61, Pro‐76, and Gly‐92. Similar residues occur in most protein disulfide isomerase sequences. Most of these residues form the surface around the active site that appears to facilitate interactions with other enzymes.

Other structurally important residues are also conserved. A proline at position 40 causes a kink in the alpha‐2 helix and thus provides the proper position of the active site residues at the amino end of this helix. Pro‐76 is important in maintaining the native structure of the molecule. In addition, residues forming the internal contact surfaces between the secondary structural elements are generally unchanged such as Phe‐12, Val‐25, and Phe‐27.
Detection of common three‐dimensional substructures in proteins
Authors: Vriend Gerrit , Sander Chris
Published: May 2005

We present a fully automatic algorithm for three‐dimensional alignment of protein structures and for the detection of common substructures and structural repeats. Given two proteins, the algorithm first identifies all pairs of structurally similar fragments and subsequently clusters into larger units pairs of fragments that are compatible in three dimensions. The detection of similar substructures is independent of insertion/deletion penalties and can be chosen to be independent of the topology of loop connections and to allow for reversal of chain direction. Using distance geometry filters and other approximations, the algorithm, implemented in the WHAT IF program, is so fast that structural comparison of a single protein with the entire database of known protein structures can be performed routinely on a workstation. The method reproduces known non‐trivial superpositions such as plastocyanin on azurin. In addition, we report surprising structural similarity between ubiquitin and a (2Fe‐2S) ferredoxin.
Sesam: A relational database for structure and sequence of macromolecules
Authors: Huysmans Martina , Richelle Jean , Wodak Shoshana J.
Published: May 2005

A system is described that provides ways of integrating data on protein structure, sequence, and survey results, with molecular graphics and molecular mechanics software. Its major component is the relational database SESAM, presently implemented under the commercial package SYBASE. By desin, the database allows full integration—within the same data organization—of raw data on protein structure, sequence, ligands, and heterogroups, obtained from the Brookhaven Protein Databank, with pure sequence information available from other databanks such as SWISS‐PROT. It contains in addition higher level descriptions of structural and topological properties, as well as survey results, obtained by executing specialized computer programs. Aside from the very useful attribute of closely combining structural and nonstructural information, other important features distinguish it from analogous systems developed elsewhere. It includes a molecular dictionary with complete description of geometric properties and energy parameters used in modeling and conformational energy calculations. Using this dictionary, structural data are validated by checking for localized inconsistencies in atomic coordinates, atomic symbols, chirality definitions, and flagging errors and incomplete entries. Because of both the dictionary and the validation procedures, SESAM can be readily interfaced with conventional molecular graphics and mechanics software packages, or with other specialized application programs. With the aid of appropriate interfaces, data access is sufficiently fast for SESAM to be interrogated interactively. Prototypes of user interfaces, as well as an interface with the molecular graphics package BRUGEL, are described and the power of the system is illustrated in applications such as homology‐based protein modeling, computer‐aided protein design, protein structure predictions, analysis of local structure motifs, and of relationships between protein sequence and structure.
Expert system for predicting protein localization sites in gram‐negative bacteria
Authors: Nakai Kenta , Kanehisa Minoru
Published: May 2005

We have developed an expert system that makes use of various kinds of knowledge organized as “if‐then” rules for predicting protein localization sites in Gram‐negative bacteria, given the amino acid sequence information alone. We considered four localization sites: the cytoplasm, the inner (cytoplasmic) membrane, the periplasm, and the outer membrane. Most rules were derived from experimental observations. For example, the rule to recognize an inner membrane protein is the presence of either a hydrophobic stretch in the predicted mature protein or an uncleavable N‐terminal signal sequence. Lipoproteins are first recognized by a consensus pattern and then assumed present at either the inner or outer membrane. These two possibilities are further discriminated by examining an acidic residue in the mature N‐terminal portion. Furthermore, we found an empirical rule that periplasmic and outer membrane proteins were successfully discriminated by their different amino acid composition. Overall, our system could predict 83% of the localization sites of proteins in our database.
Effect of urey–bradley–shimanouchi force field on the harmonic dynamics of proteins
Authors: Derreumaux Philippe , Vergoten Géarard
Published: May 2005

A normal mode analysis of bovine pancreatic trypsin inhibitor is carried out by using a Urey‐Bradley–Shimanouchi potential energy function. The density of vibrational states, the magnitudes, and time scales of the atomic fluctuations are compared with experimental and theoretical results obtained by the more commonly used potential energy functions. The atomic fluctuations of Lys‐15 are subject to extensive considerations as this residue is buried in the trypsin specificity pocket. It is found that Arg‐17 is likely to be of importance in order to understand the way BPTI binds on trypsin.
Asymmetric inclusion by de novo designed proteins: Fluorescence probe studies on amphiphilic α‐helix bundles
Authors: Morii Hisayuki , Ichimura Kunihiro , Uedaira Hatsuho
Published: May 2005

The inclusion feature and supersecondary structure of the de novo designed proteins which are constructed with several amphiphilic α‐helices and flexible linkage parts were investigated with fluorescence probes. Five types of small proteins (or peptides) have been designed, which are composed of 2, 3, 4, 4, and 6 helices, respectively, and are linked with only linear junctions except for one of 4‐helix proteins. All of these proteins have inclusion ability for hydrophobic fluorophores. Further, by the analysis of fluorescence polarization anisotropy, it was suggested that these proteins include guest molecules in compact helix bundles constructed with about 4 helices. Asymmetric inclusion of both monomer and stacked dimer of acridine orange derivatives was found by means of induced circular dichroism except for the 4‐helix protein with cross‐junction. The chirality of the included dimer proved to be in accordance with the chiral sense of α‐helical coiled‐coil. The 6‐helix protein has especially high efficiency in inclusion for any fluorophores examined in this study and brings about a significant blue‐shift of maximal emission for 8‐anilino‐1‐naphthalenesulfonate.
Crystallization and preliminary analysis of enzyme‐substrate complexes of pyruvate kinase from rabbit muscle
Authors: Schmidt‐Bäse Karen , Buchbinder Jenny L. , Reed George H. , Rayment Ivan
Published: May 2005

Pyruvate kinase from rabbit muscle has been crystallized in a form suitable for high resolution X‐ray analysis. Complexes of the enzyme with Mn2+ and either pyruvate or oxalate crystallize from solutions of polyethyleneglycol 8000 at pH 6.0. Crystals obtained from solutions of the complexes with pyruvate or oxalate appear isomorphous and belong to the triclinic space group P1. The crystals have unit cell dimensions a = 83.3(4) Å, b = 109.4(6) Å, c = 145.7(7) Å, α = 94.9°, β = 93.6°, γ = 112.2°. These crystals diffract to better than 2.4 Å resolution and are stable in the X‐ray beam for at least 20 hr. Electron paramagnetic resonance measurements on a single crystal show that Mn2+ is bound to the crystalline protein.
Collective motions in proteins: A covariance analysis of atomic fluctuations in molecular dynamics and normal mode simulations
Authors: Ichiye Toshiko , Karplus Martin
Published: May 2005

A method is described for identifying collective motions in proteins from molecular dynamics trajectories or normal mode simulations. The method makes use of the covariances of atomic positional fluctuations. It is illustrated by an analysis of the bovine pancreatic trypsin inhibitor. Comparison of the covariance and cross‐correlation matrices shows that the relative motions have many similar features in the different simulations. Many regions of the protein, especially regions of secondary structure, move in a correlated manner. Anharmonic effects, which are included in the molecular dynamics simulations but not in the normal analysis, are of some importance in determining the larger scale collective motions, but not the more local fluctuations. Comparisons of molecular dynamics simulations in the present and absence of solvent indicate that the environment is of significance for the long‐range motions.
Crystal parameters and molecular replacement of an anticholera toxin peptide complex
Authors: Shoham Menachem , Proctor Peter , Hughes Diane , Baldwin Eric T.
Published: May 2005

TE33 is an Fab fragment of a monoclonal antibody raised against a 15‐residue long peptide (CTP3), corresponding in sequence to residues 50–64 of the cholera toxin B subunit. Crystals of the complex between TE33 and CTP3 have been grown from 20% (w/v) polyethylene glycol‐8000 at pH 4.0. The crystals are orthorhombic, space group P21212, with unit cell dimensions a = 104.15, b = 110.61, and c = 40.68 Å. X‐Ray data have been collected to a resolution of 2.3 Å. The asymmetric unit contains one molecule of Fab and one molecule of CTP3. The presence of CTP3 has been demonstrated by fluorescence quenching of the dissolved crystal after X‐ray data collection. A molecular replacement solution was found based on the coordinates of DB3, an antiprogesterone Fab fragment.
Crystallization and preliminary X‐ray investigation of a recombinant form of rat catechol O‐methyltransferase
Authors: Vidgren Jukka , Tilgmann Carola , Lundström Kenneth , Liljas Anders
Published: May 2005

Rat catechol O‐methyltransferase cDNA was introduced into an E. coli expression vector pKEX14, which utilizes the inducible T7 promoter. Active and soluble recombinant catechol O‐methyltransferase was produced in bacteria and purified to electrophoretic homogeneity by chromatographic procedures. The purified enzyme has been crystallized by the method of vapor diffusion using polyethylene glycol as precipitant. The space group is P3121 or P3221 with a = b = 51.3 Å and c = 168.5 Å and one molecule in the asymmetric unit. The crystals diffract beyond 3.2 Å and are suitable for three‐dimensional X‐ray structure determination.
A single tryptic fragment of colicin E1 can form an ion channel: Stoichiometry confirms kinetics
Authors: Levinthal Françoise , Todd A. Paul , Hubbell Wayne L. , Levinthal Cyrus
Published: May 2005

The molecularity of the ion channel formed by peptide fragments of colicin has taken on particular significance since the length of the active peptide has been shown to be less than 90 amino acids and the lumen size at least 8 Å. Cell survival experiments show that killing by colicin obeys single‐hit statistics, and ion leakage rates from phospholipid vesicles are first order in colicin concentration. However, interpretation in molecular terms is generally complicated by the requirement of large numbers of colicin molecules per cell or vesicle.

We have measured the discharge of potential across membranes of small phospholipid vesicles by following the changes in binding of potential sensitive spin labeled phosphonium ions as a function of the number of colicin fragments added. Because of the sensitivity of the method, it was possible to reliably investigate the effect of colicin in a range where there was no more than 0.2 colicins per vesicle. The quantitative results of these experiments yield a direct molecular stoichiometry and demonstrate that one C‐terminal fragment of the colicin molecule per one vesicle is sufficient to induce a rapid ion flux in these vesicles. In addition, the experiments confirm earlier findings that the colicin fragments do not migrate from one vesicle to another at pH 4.5. Similar results are obtained with large unilamellar vesicles.
Information‐theoretical entropy as a measure of sequence variability
Authors: Shenkin Peter S. , Erman Batu , Mastrandrea Lucy D.
Published: May 2005

We propose the use of the information‐theoretical entropy, S = −Σpi log2 Pi, as a measure of variability at a given position in a set of aligned sequences. pi stands for the fraction of times the i‐th type appears at a position. For protein sequences, the sum has up to 20 terms, for nucleotide sequences, up to 4 terms, and for codon sequences, up to 61 terms. We compare S and VS, a related measure, in detail with VK, the traditional measure of immunoglobulin sequence variability, both in the abstract and as applied to the immunoglobulins. We conclude that S has desirable mathematical properties that VK lacks and has intuitive and statistical meanings that accord well with the notion of variability. We find that VK and the S‐based measures are highly correlated for the immunoglobulins. We show by analysis of sequence data and by means of a mathematical model that this correlation is due to a strong tendency for the frequency of occurrence of amino acid types at a given position to be log‐linear. It is not known whether the immunoglobulins are typical or atypical of protein families in this regard, nor is the origin of the observed rank‐frequency distribution obvious, although we discuss several possible etiologies.
On the helix sense of gramicidin A single channels
Authors: Koeppe Roger E. , Providence Lyndon L. , Greathouse Denise V. , Heitz Frédéric , Trudelle Yves , Purdie Neil , Andersen Olaf S.
Published: May 2005

In order to resolve whether gramicidin A channels are formed by right‐ or left‐handed β‐helices, we synthesized an optically reversed (or mirror image) analogue of gramicidin A, called gramicidin A−, to test whether it forms channels that have the same handedness as channels formed by gramicidin M− (F. Heitz et al., Biophys. J. 40:87–89, 1982). In gramicidin M− the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therfore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of gramicidin M− in dimyristoylphosphatidylcholine vesicles is consistent with a left‐handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149–160, 1986), and the CD spectra of gramicidins A and A− are essentially mirror images of each other. Based on hybrid channel experiments, gramicidin A− and M− channels are structurally equivalent, while gramicidin A and A− channels are nonequivalent, being of opposite helix sense. Gramicidin A− channels are therefore left‐handed, and natural gramicidin A channels in phospholipid bilayers are right‐handed β6.3‐helical dimers.
Crystallization and preliminary X‐ray diffraction studies of the human major histocompatibility antigen HLA‐B27
Authors: Gorga Joan C. , Madden Dean R. , Prendergast John K. , Wiley Don C. , Strominger Jack L.
Published: May 2005

The class I major histocompatibility (MHC) antigen HLA‐B27 was purified by immunoaffinity chromatography from the homozygous human B lymphoblastoid cell line LG‐2. Detergent‐soluble HLA‐B27 was cleaved with the protease papain to remove the hydrophobic transmembrane region and the cytoplasmic tail. Crystals of the resulting water‐soluble extracellular fragments were obtained in hanging drops by the vapor‐diffusion method. The crystals are triclinic, space group P1, with unit cell dimensions a = 45.9 Å, b = 71.0 Å, c = 83.7 Å, α = 79.4°, β = 88.5°, γ = 89.9°, and diffract beyond 2.5 Å resolution.
Activity of crystalline turkey egg white lysozyme
Authors: Howell P. Lynne , Warren Charles , Amatayakul‐Chantler Supavadee , Petsko Gregory A. , Hajdu Janos
Published: May 2005

Hexagonal crystals of turkey egg white lysozyme have been examined for activity in order to evaluate their potential for use in time‐resolved X‐ray crystallographic experiments. Substrates used in this study were hexa‐N‐acetylglucosamine (hexa‐GlcNAc) and a modified analogue of hexa‐GlcNAc where the terminal sugar ring was opened by reduction with tritiated sodium borohydride. This gave a labeled β‐N‐acetylglucosaminitol unit at the sixth position of the sugar chain and allowed easy quantitation of enzymatic cleavage on TLC plates. Using these substrates, it has been shown that turkey egg white lysozyme is enzymatically active in the crystal. Enzyme dispersed in the buffer surrounding the crystal does not show detectable activity under conditions relevant to an X‐ray experiment. Unmodified hexa‐GlcNAc is hydrolyzed into di‐, tri‐, and tetrasaccharides in the crystal. This cleavage pattern is different from that obtained with hen egg white lysozyme in solution and likely causes of the differences are discussed. The reduced radiolabeled oligosaccharide has a unique cleavage pattern with trisaccharides as the products. The specific activity of the enzyme with the radiolabelled analogue was 9.8 (± 1.0) × 10−7 mmol/min/mg protein at 22°C in the crystal.
Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate
Authors: Kiefhaber Thomas , Grunert Hans‐Peter , Hahn Ulrich , Schmid Franz X.
Published: May 2005

The replacement of tryptophan 59 of ribonuclease T1 by a tyrosine residue does not change the stability of the protein. However, it leads to a strong acceleration of a major, proline‐limited reaction that is unusually slow in the refolding of the wild‐type protein. The distribution of fast‐ and slow‐folding species and the kinetic mechanism of slow folding are not changed by the mutation. Trp‐59 is in close contact to Pro‐39 in native RNase T1 and probably also in an intermediate that forms rapidly during folding. We suggest that this specific interaction interferes with the trans→cis reisomerization of the Tyr‐38–Pro‐39 bond at the stage of a native‐like folding intermediate. The steric hindrance is abolished either by changing Trp‐59 to a less bulky residue, such as tyrosine, or, by a destabilization of folding intermediates at increased concentrations of denaturant. Under such conditions folding of the wild‐type protein and of the W59Y variant no longer differ. These results provide strong support for the proposal that trans→cis isomerization of Pro‐39 is responsible for the major, very slow refolding reaction of RNase T1. They also indicate that specific tertiary interactions in folding intermediates do exist, but do not necessarily facilitate folding. They can have adverse effects and decelerate ratelimiting steps by trapping partially folded structures.
Substrate mobility in a deeply buried active site: Analysis of norcamphor bound to cytochrome P‐450cam as determined by a 201‐psec molecular dynamics simulation
Authors: Bass Michael B. , Paulsen Mark D. , Ornstein Rick L.
Published: May 2005

While cytochrome P‐450cam, catalyzes the hydroxylation of camphor to 5‐exo‐hydroxycamphor with 100% stereospecificity, norcamphor is hydroxylated by this enzyme yielding 45% 5‐exo‐, 47% 6‐exo‐, and 8% 3‐exo‐hydroxynorcamphor (Atkins, W.M., Sligar, S.G., J. Am. Chem. Soc. 109:3754–3760, 1987). The present study describes a 201‐psec molecular dynamics (MD) simulation of norcamphorbound cytochrome P‐450cam to elucidate the relationship between substrate conformational mobility and formation of alternative products. First, these data suggest that the product specificity is, at least partially, due to the mobility of the substrate within the active site. Second, the high mobility of norcamphor in the active site leads to an average increase in separation between the home iron and the substrate of about 1.0 Å; this increase in separation may be the cause of the uncoupling of electron transfer when norcamphor is the substrate. Third, the active site water located in the norcamphor‐bound crystal structure possesses mobility that correlates well with the spin‐state equilibrium of this enzyme–substrate complex. © 1992 Wiley‐Liss, Inc.
Molecular modeling of an antigenic complex between a viral peptide and a class I major histocompatibility glycoprotein
Authors: Rognan Didier , Reddehase Matthias J. , Koszinowski Ulrich H. , Folkers Gerd
Published: May 2005

Computer simulation of the conformations of short antigenic peptides (5–10 residues) either free or bound to their receptor, the major histocompatibility complex (MHC)‐encoded glycoprotein H‐2 Ld, was employed to explain experimentally determined differences in the antigenic activities within a set of related peptides. Starting for each sequence from the most probable conformations disclosed by a pattern‐recognition technique, several energy‐minimized structures were subjected to molecular dynamics simulations (MD) either in vacuo or solvated by water molecules. Notably, antigenic potencies were found to correlate to the peptides propensity to form and maintain an overall α‐helical conformation through regular i,i+4 hydrogen bonds. Accordingly, less active or inactive peptides showed a strong tendency to form i,i+3 hydrogen bonds at their N‐terminal end. Experimental data documented that the C‐terminal residue is critical for interaction of the peptide with H‐2 Ld. This finding could be satisfactorily explained by a 3‐D Q.S.A.R. analysis postulating interactions between ligand and receptor by hydrophobic forces. A 3‐D model is proposed for the complex between a high‐affinity nonapeptide and the H‐2 Ld receptor. First, the H‐2 Ld molecule was built from X‐ray coordinates of two homologous proteins: HLA‐A2 and HLA‐Aw68, energy‐minimized and studied by MD simulations. With HLA‐A2 as template, the only realistic simulation was achieved for a solvated model with minor deviations of the MD mean structure from the X‐ray conformation. Water simulation of the H‐2 Ld protein in complex with the antigenic nonapeptide was then achieved with the template‐derived optimal parameters. The bound peptide retains mainly its α‐helical conformation and binds to hydrophobic residues of H‐2 Ld that correspond to highly polymorphic positions of MHC proteins. The orientation of the nonapeptide in the binding cleft is in accordance with the experimentally determined distribution of its MHC receptor‐binding residues (agretope residues). Thus, computer simulation was successfully employed to explain functional data and predicts α‐helical conformation for the bound peptide. © 1992 Wiley‐Liss, Inc.
Molecular mechanics simulations of a conformational rearrangement of D‐xylose in the active site of D‐xylose isomerase
Authors: Smart Oliver S. , Akins John , Blow David M.
Published: May 2005

A proposed reaction mechanism for the enzyme D‐xylose isomerase involves the ring opening of the cyclic substrate with a subsequent conformational rearrangement to an extended open‐chain form. Restrained energy minimization was used to simulate the rearrangement. In the ring‐opening step, the substrate energy function was gradually altered from a cyclic to an open‐chain form, with energy minimization after each change. The protein/sugar contact energy did not increase significantly during the process, showing that there was no steric hindrance to ring opening. The conformational rearrangement involves an alteration in the coordination of the substrate to metal ion [1], which was induced by gradually changing restraints on metal/ligand distances. By allowing varying amounts of flexibility in the protein and examining a simplified model system, the interactions of the sugar with metal ion [1] and its immediate ligands were found to be the most important contributors to the energy barrier for the change. Only small changes in the positions of protein atoms were required. The energy barrier to the rearrangement was estimated to be less than the Arrhenius activation energy for the enzymatic reaction. This is in accordance with experimental indications that the isomerization step is rate determining. © 1992 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray diffraction analysis of staphylococcal enterotoxin type C
Authors: Bohach Gregory A. , Chi Young‐In , Stauffacher Cynthia V.
Published: May 2005

The Type C staphylococcal enterotoxin produced by Staphylococcus aureus strain FRI‐909 has been crystallized using a combination of two precipitants, ammonium sulfate and polyethylene glycol 400, with the addition of small amounts of detergent. Two related crystal forms have been obtained, one triclinic, and one tetragonal, both with one toxin molecule per asymmetric unit. These crystals are stable for at least 75 hr in the X‐ray beam and diffract to at least 2.2 and 2.6 Å, respectively. The triclinic crystals have unit cell parameters a = 38.5 Å, b = 43.7 Å, c = 36.9 Å, and interaxial angles α = 99.9°, β = 95.8°, and γ = 98.5°. The tetragonal crystals are of space group P4122 with unit cell parameters a = 43.4 Å and c = 278.0 Å. © 1992 Wiley‐Liss, Inc.
Detection of local structures in reduced unfolded bovine pancreatic trypsin inhibitor
Authors: Amir Dan , Krausz Sara , Haas Elisha
Published: May 2005

The structure of BPTI and reduced BPTI in concentrated guanidinium HCI (GUHCl) in the presence of glycerol has been probed by measurements of dynamic nonradiative excitation energy transfer between probes attached to its amino groups. Inter probe distance distributions were obtained from analysis of donor fluorescence decay curves and used to characterize local structures in unordered states of the protein. Site specifically fluorescently labeled BPTI derivatives (1‐n)BPTI (n = 15, 20, 41, 46) were used, each carrying a 2‐methoxy‐naphthyl‐1‐methylenyl group (MNA) at the N‐terminal amino group of arg1 and 7‐(dimethylamino)‐coumarin‐4‐yl‐acetyl residue (DA‐coum) at one of its ε‐NH2 groups of the lysine side chains. Analysis of donor fluorescence decay kinetics gave the interprobe distance distributions in the native and denatured states.

The N‐terminal‐segment, residues 1–15, is in an extended conformation (with an average interprobe distance of 34 ± 2 Å) in the native state. Upon unfolding by reduction with DTT or β‐mercapto ethanol in 6 M GUHCl/glycerol mixture, the conformation of this segment relaxed to a state characterized by a reduced averageinterprobe distance and a larger width of the distances distribution. The average distance between residues 1 and 26, i.e., between the N‐terminus and the turn of the twisted β sheet element (residues 18–35), increased upon unfolding. At −30°C in the above solvent, the distribution between these two sites was probably composed of two conformational subpopulations. About 45 ±20% of the molecules were characterized by a short interprobe distance (like the native state) representing a compact conformation, and 55 ± 20% of the molecules showed large interprobe distances representing an expanded (unfolded) conformation.

Thus local structures seem to exist in reduced denatured BPTI even underdenaturing conditions in 6 M GUHCl/glycerol mixtures. Some of those structures are unstable in guanidinium isothiocyanate (GUSCN). The method introduced here is suitable for probing local structures and very long range interactions in unfolded folded proteins and for search for folding initiation sites (FISs) and early folding intermediates. © 1992 Wiley‐Liss, Inc.
Molecular dynamics of HIV‐1 protease
Authors: Harte William E. , Swaminathan S. , Beveridge David L.
Published: May 2005

Molecular dynamics simulations have been carried out based on the GROMOS force field on the aspartyl protease (PR) of the human immunodeficiency virus HIV‐1. The principal simulation treats the HIV‐1 PR dimer and 6990 water molecules in a hexagonal prism cell under periodic bundary conditions and was carried out for a trajectory of 100 psec. Corresponding in vacuo simulations, i.e., treating the isolated protein without solovent, were carried out to study the influence of solvent on the simulation. The results indicate that including waters explicitly in the simlation results in a model considerably closer to the crystal structure than when solvent is neglected. Detailed conformational and helicoidal analysis was perfomed on the solvated form to determine the exact nature of the dynamical model and the exact points of agreement and disagreement with the crystal structure. The calculated dynamical model was furthr elucidated by means of studies of the time evolution of the cross‐correlation coefficients for atomic displacements of the atoms comprising the protein backbone. The cross‐correlation analysis revealed significant aspects of structure originating uniquely in the dynamical motions of the molecule. In particular, an unanticipated troughspace, domain‐domain correlation was found between the mobile flap region covering the active site and a remote regions of the structure, which collectively act somewhat like a molecular cantilever. The significance of these results is discussed with respect to the inactivation of the protease by site‐specific mutagenesis, andin the design of inhibitors. © 1992 Wiley‐Liss, Inc.
NMR docking of a substrate into the X‐ray structure of staphylococcal nuclease
Authors: Weber David J. , Gittis Apostolos G. , Mullen Gregory P. , Abeygunawardana Chitrananda , Lattman Eaton E. , Mildvan Albert S.
Published: May 2005

The conformation of the staphylococcal nuclease‐bound metal–dTdA complex, previously determined by NMR methods [Weber, D.J., Mullen, G.P., Mildvan, A.S. (1991) Biochemistry 30:7425–7437] was docked into the X‐ray structure of the enzyme–Ca2+‐3′,5′‐pdTp complex [Loll, P.J., Lattman, E.E. (1989) Proteins: Struct., Funct., Genet. 5:183–201] by superimposing the metal ions, taking into account intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr‐85, Tyr‐113, and Tyr‐115 to proton resonances of the leaving dA moiety of dTdA, and energy minimization to relieve small overlaps. The proton resonances of the Phe, Tyr, and Trp residues of the enzyme in the ternary enzyme–La3+‐dTdA complex were sequence specifically assigned by 2D phase‐sensitive NOESY, with and without deuteration of the aromatic protons of the Tyr residues, and by 2D heteronu‐clear multiple quantum correlation (HMQC) spectroscopy and 3D NOESY‐HMQC spectros‐copy with 15N labeling. While resonances of most Phe, Tyr and Trp residues were unshifted by the substrate dTdA from those found in the enzyme–La3+–3′,5′‐pdTp complex and the enzyme–Ca2+–3′,5′‐pdTp complex, proton resonances of Tyr‐85, Tyr‐113, Tyr‐115, and Phe‐34 were shifted by 0.08 to 0.33 ppm and the 15N resonance of Tyr‐113 was shifted by 2.1 ppm by the presence of substrate. The optimized position of enzyme‐bound dTdA shows the 5′‐dA leaving group to partially overlap the inhibitor, 3′,5′‐pdTp (in the X‐ray structure). Tne 3′‐TMP moiety of dTdA points toward the solvent in a channel defined by Ile‐18, Asp‐19, Thr‐22, Lys‐45, and His‐46. The phosphate of dTdA is coordinated by the metal, and an adjacent inner sphere water ligand is positioned to donate a hydrogen bond to the general base Glu‐43 and to attack the phosphorus with inversion. Arg‐35 and Arg‐87 donate monodentate hydrogen bonds to different phosphate oxygens of dTdA, with Arg‐87 positioned to protonate the leaving 5′‐oxygen of dA, thus clarifying the mechanism of hydrolysis. Model building of an additional 5′‐dGMP onto the 3′‐oxygen of dA placed this third nucleotide onto a surface cleft near residues Glu‐80, Asp‐83, Lys‐84, and Tyr‐115 with its 3′‐OH group accessible to the solvent, thus defining the size of the substrate binding site as accommodating a trinucleotide. © 1992 Wiley‐Liss, Inc.
NMR restraint analysis of transforming growth factor α: A key component for NMR structure refinement
Authors: Brown Frank K. , Hempel Judith C. , Jeffs Peter W.
Published: May 2005

Structures of the protein, transforming growth factor α (TGF‐α), have been derived from NMR data using distance geometry and subsequent energy refinement. Analysis of the sequential NOE distance bounds using a template algorithm provides a check for consistency in the calculation of bounds, stereospecific assignment of prochiral centers, and secondary structure assignment. Application of the template algorithm to the long range NOEs found within the NMR data sets collected at pH 6.3 and pH 3.4 is used to assess the confidence levels for the accuracy of the structures obtained from modeling. The method also provides critical insight in differentiating regions of the structure that are well defined from those that are not. Use of the restraint analysis protocol is shown to be a powerful adjunct to currently used methods for the assignment of protein structures from NMR data. © 1992 Wiley‐Liss, Inc.
Alkaline phosphatase‐somatostatin hybrid proteins as probes for somatostatin‐14 receptors
Authors: Langen Hanno T. , Taylor John W.
Published: May 2005

By inserting appropriate peptide ligands into surface loops on globular proteins, we expect to develop probes for the location, accessibility, and steric and electrostatic environment of these ligand‐binding sites on their membrane‐bound receptors. Three residues in a loop on the surface of E. coli alkaline phosphatase were substituted by an 18‐residue peptide containing the receptor‐binding segment of somatostatin‐14 without significantly affecting the catalytic properties of the enzyme. This hybrid protein was then used to investigate the ligand‐binding site of somatostatin receptors. Tryptic cleavage of the hybrid protein within the inserted sequence, and binding of the hybrid protein to antisomatostatin antibodies demonstrated the surface accessibility of the guest peptide. Both the wild‐type enzyme and the hormone‐enzyme hybrid displaced 125I‐labeled somatostatin from rat brain membrane receptors only at high concentrations. How‐ever, chemical cationization of the hybrid protein, which again did not disturb the phosphatase activity, enhanced its receptor‐binding potency to a level only 23 times lower than that of somatostatin itself and 280 times higher than that of the cationized wild‐type protein. This alkaline phosphatase/somatostatin hybrid protein appears, therefore, to be a suitable starting point for the development of probes for the steric and electrostatic environment of the ligand‐binding site of somatostatin receptors. © 1992 Wiley‐Liss, Inc.
Probing β‐lactamase structure and function using random replacement mutagenesis
Authors: Palzkill Timothy , Botstein David
Published: May 2005

A new analytical mutagenesis technique is described that involves randomining the DNA sequence of a short stretch of a gene (3–6 codons) and determining the percentage of all possible random sequences that produce a functional protein. A low percentage of functional random sequences in a complete library of random substitutions indicates that the region mutagenized is important for the structure and/or function of the protein. Repeating the mutagenesis over many regions throughout a protein gives a global perspective of which amino acid sequences in a protein are critical. We applied this method to 66 codons of the gene encoding TEM‐1 β‐lactamase in 19 separate experiments. We found that TEM‐1 β‐lactamase is extremely tolerant of amino acid substitutions: on average, 44% of all mutants with random substitutions function and 20% of the substitutions are expressed, secreted, and fold well enough to function at levels similar to those for the wild‐type enzyme. We also found a few exceptional regions where only a few random sequences function. Examination of the X‐ray structures of homologous β‐lactamases indicates that the regions most sensitive to substitution are in the vicinity of the active site pocket or buried in the hydrophobic core of the protein. DNA sequence analysis of functional random sequences has been used to obtain more detailed information about the amino acid sequence requirements for several regions and this information has been compared to sequence conservation among several related β‐lactamases. © 1992 Wiley‐Liss, Inc.
Empirical solvation models in the context of conformational energy searches: Application to bovine pancreatic trypsin inhibitor
Authors: Williams Roger L. , Vila Jorge , Perrot Georges , Scheraga Harold A.
Published: May 2005

Continuum solvation models that estimate free energies of solvation as a function of solvent accessible surface area are computationally simple enough to be useful for predicting protein conformation. The behaviour of three such solvation models has been examined by applying them to the minimization of the conformational energy of bovine pancreatic trypsin inhibitor.

The model differ only with regard to how the constants of proportionality between free energy and surface area were derived. Each model was derived by fitting to experimentally measured equilibrium solution properties. For two models, the solution property was free energy of hydration. For the thrid, the property was NMR coupling constants. The purpose of this study is to determine the effect of applying these solvation models to the nonequilibrium conformations of a protein arising in the course of global searches for conformational energy minima. Two approaches were used: (1) local energy minimization of an ensemble of conformations similar to the equilibrium conformation and (2) global search trajectories using Monte Carlo plus minimization starting from a single conformation similar to the equilibrium conformation.

For the two models derived from free energy measurements, it was found that both the global searches and local minimizations yielded conformations more similar to the X‐ray crystallographic structures than did searches or local minimizations carried out in the absence of a solvation component of the conformational energy. The model derived from NMR coupling constants behaved similarly to the other models in the context of a global search trajectory. For one of the models derived from measured free energies of hydration, it was found that minimization of an ensemble of near‐equilibrium conformations yielded a new ensemble in which the conformation most similar to the X‐ray determined structure PTI4 had the lowest total free energy.

Despite the simplicity of the continuum slvation models, the final conformation generated in the trajectories for each of the models exhibited some of the characteristics that have been reported for conformations obtained from molecular dynamics simulations in the presence of a bath of explicit water molecules. They have smaller root mean square (rms) deviations from the experimentally determined conformation, fewer incorrect hydrogen bonds, and slightly larger radii of gyration than do conformations derived from search trajectories carried out in the absence of sovlent. © 1992 Wiley‐Liss, Inc.
Calmodulin binding to α1‐purothionin: Soloution binding and modeling of the complex
Authors: Rao Usha , Teeter Martha M. , Erickson‐Viitanen Susan , DeGrado William F.
Published: May 2005

CD and fluorescence spectroscopic measurements show that calmodulin (CaM) binds to purothionins (alpha;1‐purothionin: α1‐PT; β‐purothionin: β‐PT) in 1:1 stoichiometry with an affinity similar to that exhibited with the tightest binding CaM‐binding peptides. Using the available crystal structures of CaM and α1‐PT, a model has been built for the interaction of CaM and α1‐PT and subjected to potential energy minimization. In the model, there is a bend in the central helix of CaM similar to that suggested by Persechini and Kretsinger (J. Card. Pharm. 12:501–512, 1988). α1‐PT fits snugly into the cavity formed by the bent CaM molecule with each of its two helices making apolar interactions with each of the two hydrophobic clefts situated at the terminal domains of CaM. The complex is further stabilized by numerous polar and electrostatic interactions on the rims of the clefts. Our model is compared with two other similar models previously re‐ported for the CaM complexes with other helical peptides and generalizations about the mode of CaM binding to target proteins are made, which have wide relevance to the function of CaM. By analogy, a similar model is pre‐dicted for a CaM–β‐PT complex. © 1992 Wiley‐Liss, Inc.
Crystal structure of the reduced form of p‐ hydroxybenzoate hydroxylase refined at 2.3 Å resolution
Authors: Schreuder Herman A. , van der Laan Jan M. , Swarte Myra B. A. , Kalk Kor H. , Hol Wim G. J. , Drenth Jan
Published: May 2005

The crystal structure of the reduced form of the enzyme p‐hydroxybenzoate hydroxylase from Pseudomonasm fluorescens, complexed with its substrate p‐hydroxybenzoate, has been obtained by protein X‐ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzyme‐substrate complex in deaerated mother liquor containing 300–400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme‐bound FAD by NADPH. This was confirmed by single crystal spectroscopy.

X‐ray data to 2.3 Å were collected on oscillation films using a rotating anode generator as an X‐ray source. After data processing and reduction, restrained least squares refinement using the 1.9 Å structure of the oxidized enzyme‐substrate complex as a starting model, yielded a crystallographic R‐factor of 14.8% for 11,394 reflections. The final model of the reduced complex contains 3,098 protein atoms, the FAD molecule, the substrate p‐hydroxybenzoate and 322 solvent molecules.

The structures of the oxidized and reduced forms of the enzyme‐substrate complex were found to be very similar. The root‐mean‐square discrepancy for all atoms between both structures was 0.38 Å. The flavin ring is almost completely planar in the final model, although it was allowed to bend or twist during refinement. The observed angle between the benzene and the pyrimidine ring is 2° This value should be compared with observed values of 10° for the oxidized enzyme‐substrate complex and 19° for the enzyme‐product complex. The position of the substrate is virtually unaltered with respect to its position in the oxidized enzyme. No trace of a bound NADP+ or NADPH molecule was found. © 1992 Wiley‐Liss, Inc.
Effects of changing the interaction between subdomains on the thermostability of Bacillus neutral proteases
Authors: Eijsink Vincent G. H. , Vriend Gerrit , van der Vinne Bernard , Hazes Bart , van den Burg Bertus , Venema Gerard
Published: May 2005

Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr‐sub) and the thermostable neutral protease of B. stearothermophilus (Npr‐ste) were produced by means of site‐directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to alter the interaction between the middle and C‐terminal subdomain of these enzymes. In all Nprs a cluster of hydrophobic contacts centered around residue 315 contributes to this interaction. In thermostable Nprs (like Npr‐ste) a 10 residue β‐hairpin, covering the domain interface, makes an additional contribution. The hydrophobic residue at position 315 was replaced by smaller amino acids. In addition, the β‐hairpin was deleted from Npr‐ste and inserted into Npr‐sub. The changes in thermostability observed after these mutations confirmed the importance of the hydrophobic cluster and of the β‐hairpin for the structural integrity of Nprs. Combined mutants showed that the effects of individual mutations affecting the inter action between the subdomains were not additive. The effects on thermostability decreased as the strength of the subdomain interaction increased. The results show that once the subdomain interface is sufficiently stabilized, additional stabilizing mutations at the same interface do not further increase thermostability. The results are interpreted on the basis of a model for the thermal inactivation of neutral proteases, in which it is assumed that inactivation results from the occurrence of local unfolding processes that render these enzymes susceptible to autolysis. © 1992 Wiley‐Liss, Inc.
Hydrogen exchange in native and denatured states of hen egg‐white lysozyme
Authors: Radford Sheena E. , Buck Matthias , Topping Karen D. , Dobson Christopher M. , Evans Philip A.
Published: May 2005

The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30°C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 105–107 times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35°C, by lowering the stability of the native state through selective cleavage of the Cys‐6–Cys‐127 disulfide crosslink (CM6−127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20‐fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native‐like structure do not persist significantly in the denatured state of CM6−127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69°C, resembles that of the acid‐denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme. © 1992 Wiley‐Liss, Inc.