bluuebunny/crossref_metadata_2025_split huggingface dataset:
Prediction of titration properties of structures of a protein derived from molecular dynamics trajectories
Authors: Wlodek S.T. , Antosiewicz J. , Mccammon J.A. 
Published: July 2010

AbstractThis paper explores the dependence of the molecular dynamics (MD) trajectory of a protein molecule on the titration state assigned to the molecule. Four 100‐ps MD trajectories of bovine pancreatic trypsin inhibitor (BPTI) were generated, starting from two different structures, each of which was held in two different charge states. The two starting structures were the X‐ray crystal structure and one of the solution structures determined by NMR, and the charge states differed only in the ionization state of N terminus.Although it is evident that the MD simulations were too short to sample fully the equilibrium distribution of structures in each case, standard Poisson‐Boltzmann titration state analysis of the resulting configurations shows general agreement between the overall titration behavior of the protein and the charge state assumed during MD simulation: at pH 7, the total net charge of the protein resulting from the titration analysis is consistently lower for the protein with the N terminus assumed to be neutral than for the protein with the N terminus assumed to be charged.For most of the ionizable residues, the differences in the calculated pKαs among the four trajectories are statistically negligible and remain in good agreement with the data obtained by crystal structure titration and by experiment. The exceptions include the N terminus, which responds directly to the change of its imposed charge; the C terminus, which in the NMR structure interacts strongly with the former; and a few other residues (Arg 1, Glu 7, Tyr 35, and Arg 42) whose pKαs reflect the initial structure and the limited trajectory lengths. This study illustrates the importance of the careful assignment of protonation states at the start of MD simulations and points to the need for simulation methods that allow for the variation of the protonation state in the calculation of equilibrium properties.
Complete 1H, 13C, and 15N NMR resonance assignments and secondary structure of human glutaredoxin in the fully reduced form
Authors: Sun Chaohong , Bushweller John H. , Holmgren Arne 
Published: July 2010

AbstractHuman glutaredoxin is a member of the glutaredoxin family, which is characterized by a glutathione binding site and a redox‐active dithiol/disulfide in the active site. Unlike Escherichia coli glutaredoxin‐1, this protein has additional cysteine residues that have been suggested to play a regulatory role in its activity. Human glutaredoxin (106 amino acid residues, Mr = 12,000) has been purified from a pET expression vector with both uniform 15N labeling and 13C/15N double labeling. The combination of three‐dimensional 15N‐edited TOCSY, 15N‐edited NOESY, HNCA, HN(CO)CA, and gradient sensitivity‐enhanced HNCACB and HNCO spectra were used to obtain sequential assignments for residues 2‐106 of the protein. The gradient‐enhanced version of the HCCH‐TOCSY pulse sequence and HCCH‐COSY were used to obtain side chain 1H and 13C assignments. The secondary structural elements in the reduced protein were identified based on NOE information, amide proton exchange data, and chemical shift index data. Human glutaredoxin contains five helices extending approximately from residues 4‐10, 24‐36, 53‐64, 83‐92, and 94‐104. The secondary structure also shows four β‐strands comprised of residues 15‐19, 43‐48, 71‐75, 78‐80, which form a β‐sheet almost identical to that found in E. coli glutaredoxin‐1. Complete 1H, 13C, and 15N assignments and the secondary structure of fully reduced human glutaredoxin are presented. Comparison to the structures of other glutaredoxins is presented and differences in the secondary structure elements are discussed.
Conformational stability of pGEX‐expressed Schistosoma japonicum glutathione S‐transferase: A detoxification enzyme and fusion‐protein affinity tag
Authors: Kaplan Warren , Erhardt Julija , Sluis‐Cremer Nicolas , Dirr Heini , Hüsler Paul , Klump Horst 
Published: July 2010

AbstractA glutathione S‐transferase (SJ26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX‐2T plasmid and is used widely as a fusion‐protein affinity tag. It contains all 217 residues of SJ26GST and an additional 9‐residue peptide linker with a thrombin cleavage site at its C‐terminus. Size‐exclusion HPLC (SEC‐HPLC) and SDS‐PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160‐kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea‐ and temperature‐induced unfolding techniques. Fluorescence‐spectroscopy, SEC‐HPLC, urea‐ and temperature‐gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two‐state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a ΔG°(H2O) = 26.0 ± 1.7 kcal/mol. The strong relationship observed between the m‐value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two‐state unfolding transition model with values of ΔCP = 7,440 J/mol per K, ΔH = 950.4 kj/mol, and ΔS = 1,484 J/mol.
Structural characterization of the molten globule of α‐lactalbumin by solution X‐ray scattering
Authors: Kataoka Mikio , Tokunaga Fumio , Kuwajima Kunihiro , Goto Yuji 
Published: July 2010

AbstractA compact denatured state is often observed under a mild denaturation condition for various proteins. A typical example is the α‐lactalbumin molten globule. Although the molecular compactness and shape are the essential properties for defining the molten globule, there have been ambiguities of these properties for the molten globule of α‐lactalbumin. Using solution X‐ray scattering, we have examined the structural properties of two types of molten globule of α‐lactalbumin, the apo‐protein at neutral pH and the acid molten globule. The radius of gyration for the native holo‐protein was 15.7 Å, but the two different molten globules both had a radius of gyration of 17.2 Å. The maximum dimension of the molecule was also increased from 50 Å for the native state to 60 Å for the molten globule. These values clearly indicate that the molten globule is not as compact as the native state. The increment in the radius of gyration was less than 10% for the α‐lactalbumin molten globule, compared with up to 30% for the molten globules of other globular proteins. Intramolecular disulfide bonds restrict the molecular expansion of the molten globule. The distance distribution function of the α‐lactalbumin molten globule is composed of a single peak suggesting a globular shape, which is simply swollen from the native state. The scattering profile in the high Q region of the molten globule indicates the presence of a significant amount of tertiary fold. Based on the structural properties obtained by solution X‐ray scattering, general and conceptual structural images for the molten globules of various proteins are described and compared with the individual, detailed structural model obtained by nuclear magnetic resonance.
GroEL‐Mediated protein folding
Authors: Fenton Wayne A. , Horwich Arthur L. 
Published: July 2010

Abstract
Architecture of GroEL and GroES and the reaction pathway
Architecture of the chaperonins
Reaction pathway of GroEL‐GroES‐mediated folding

Polypeptide binding
A parallel network of chaperones binding polypeptides in vivo
Polypeptide binding in vitro
Role of hydrophobicity in recognition
Homologous proteins with differing recognition—differences in primary structure versus effects on folding pathway
Conformations recognized by GroEL
Refolding studies
Binding of metastable intermediates
Conformations while stably bound at GroEL

Binding constants and rates of association
Conformational changes in the substrate protein associated with binding by GroEL
Observations
Kinetic versus thermodynamic action of GroEL in mediating unfolding
Crossing the energy landscape in the presence of GroEL

ATP binding and hydrolysis—driving the reaction cycle
GroEL‐GroES‐polypeptide ternary complexes—the folding‐active cis complex
Cis and trans ternary complexes
Symmetric complexes
The folding‐active intermediate of a chaperonin reaction—cis ternary complex
The role of the cis space in the folding reaction
Folding governed by a “timer” mechanism
Release of nonnative polypeptides during the GroEL‐GroES reaction
Release of both native and nonnative forms under physiologic conditions
A role for ATP binding, as well as hydrolysis, in the folding cycle

Concluding remarks
Identification of transmembrane tryptic peptides of rhodopsin using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry
Authors: Barnidge David R. , Dratz Edward A. , Sunner Jan , Jesaitis Algirdas J. 
Published: July 2010

AbstractThe application of mass spectrometry for determining the topography of integral membrane proteins has focused primarily on the mass determination of fragments that do not reside in the lipid bilayer. In this work, we present the accurate mass determination of transmembrane tryptic peptides of bovine rhodopsin using matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. The ability to determine the accurate mass of hydrophobic transmembrane peptides will facilitate the mapping of ligand binding sites in membrane receptors. It will also augment the determination of membrane spanning regions from integral membrane proteins digested in lipid bilayers. Affinity‐purified rhodopsin in detergent and rhodopsin in retinal rod membranes were digested with trypsin. Tryptic peptides were separated using reverse‐phase, high‐performance liquid chromatography at 55°C with the detergent octyl‐β‐glucoside in the mobile phase. Four of the six transmembrane tryptic peptides of rhodopsin were identified, ranging in mass from 3,260 Da to 6,528 Da. The identities of the peptides were confirmed by Edman microsequencing. In addition, heterogeneity in the glycosylation of the N‐terminal tryptic peptide of rhodopsin was identified by MALDI MS, without modifying the carbohydrate prior to analysis.
Metal‐ and DNA‐binding properties and mutational analysis of the transcription activating factor, B, of coliphage 186: A prokaryotic C4 zinc‐finger protein
Authors: Pountney Dean L. , Tiwari Ravi P. , Egan J. Barry 
Published: July 2010

AbstractColiphage 186 B is a 72‐amino acid protein belonging to the Ogr family of analogous transcription factors present in P2‐like phage, which contain a Cys‐X2‐Cys‐X22‐Cys‐X4‐Cys presumptive zinc‐finger motif. The molecular character‐ ization of these proteins has been hampered by their insolubility, a difficulty overcome in the present study by obtaining B as a soluble cadmium‐containing derivative (CdB). Atomic absorption spectroscopy showed the presence of one atom of cadmium per molecule of purified CdB. The UV absorption spectrum revealed a shoulder at 250 nm, characteristic of CysS‐Cd(II) ligand‐to‐metal charge‐transfer transitions, and the difference absorption coefficient after acidification (Δϵ248, 24 mM−1 cm−1) indicated the presence of a Cd(Cys‐S), center. Gel mobility shift analysis of CdB with a 186 late promoter demonstrated specific DNA‐binding (KD,app 3‐4 μM) and the protein was shown to activate transcription in vitro from a promoter‐reporter plasmid construct. The B DNA‐binding site was mapped by gel shift and DNAase I cleavage protection experiments to an area between −70 and −43 relative to the transcription start site, coincident with the consensus sequence, GTTGT‐N8‐TNANCCA, from −66 to −47 of the 186 and P2 late promoters. Inactive B point mutants were obtained in the putative DNA‐binding loop of the N‐terminal zinc‐finger motifandin a central region thought to interact with the Escherichia coli RNA polymerase a‐subunit. A truncated B mutant comprising the first 53 amino acids (Bl‐53) exhibited close to wild‐type activity, showed a DNA‐binding affinity similar to that of the full‐length protein, and could be reconstituted with either Cd or Zn. Gel permeation analysis revealed that B 1‐53 was a majority dimeric species whereas wild‐type B showed larger oligomers. 186 B therefore exhibitsa potentially linear organization of functional regions comprising an N‐terminal C4 zinc‐finger DNA‐binding region, a dispensable C‐terminal region involved in protein self‐association, and a central region that interacts with RNA polymerase.
Improvement of diffraction quality upon rehydration of dehydrated icosahedral enterococcus faecalis pyruvate dehydrogenase core crystals
Authors: Izard Tina , Sarfaty Steve , Westphal Adrie , De Kok Arie , Hol WIM G. J. 
Published: July 2010

AbstractMembers of the family of 2‐oxoacid dehydrogenase multienzyme complexes catalyze the oxidative decarboxylation of α‐keto acids and are among the most remarkable enzymatic machineries in the living cell. These multienzyme complexes combine a highly symmetric (cubic or icosahedral) core with a dynamic and flexible arrangement of numerous subunits and domains surrounding the core. The center of the complex is formed by either 24 or 60 copies of dihydrolipoamide acetyltransferase (E2)–a multidomain enzyme. The hollow icosahedral cores are composed of 60 identical subunits of the catalytic domain of E2 with a molecular weight of about 1.8 million Da. Bipyramidal crystals suitable for X‐ray diffraction of the icosahedral core of the pyruvate dehydrogenase multienzyme complex from Enterococcus faecalis were grown up to 0.7 mm in each dimension. The crystals belong to space group R32 with a = b = 244.3 Å and c = 920.9 Å (hexagonal setting), and have a solvent content of 73%. The asymmetric unit contains one‐third of the molecule, i.e., 20 of the 60 subunits. Initial X‐ray crystallographic data to 7 Å resolution were collected at cryotemperatures at synchrotron facilities. Interestingly, the diffraction was improved significantly upon rehydrating dehydrated crystals and extended to 4.2 Å.
Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex
Authors: Linnevers Christopher J. , Mcgrath Mary E. , Armstrong Andy , Mistry Firoz R. , Barnes Michael G. , Klaus Jeffrey L. , Palmer James T. , Katz Bradley A. , Brömme Dieter 
Published: July 2010

AbstractCathepsin K is a cysteine protease of the papain family, which is predominantly expressed in osteoclasts, and is regarded as a key protease in bone remodeling. To facilitate structural studies of the protein, the wild‐type sequence of the protease has been mutated so as to replace a potential N‐glycosylation site. We have expressed the mutant human cathepsin K to 190 mg/5 L using the Pichia pastoris expression system. Cathepsin K was inactivated with the mechanism‐based inhibitor, APC3328, and crystallized from magnesium formate. A 2.2 Å X‐ray data set has been collected on crystals belonging to space group P212121, with a = 41.66 Å, b = 51.41 Å, and c = 107.72 Å. There is most likely one molecule per asymmetric unit.
Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: The current state of affairs
Authors: Mewies Martin , McIntire William S. , Scrutton Nigel S. 
Published: July 2010

AbstractThe first identified covalent flavoprotein, a component of mammalian succinate dehydrogenase, was reported 42 years ago. Since that time, more than 20 covalent flavoenzymes have been described, each possessing one of five modes of FAD or FMN linkage to protein. Despite the early identification of covalent flavoproteins, the mechanisms of covalent bond formation and the roles of the covalent links are only recently being appreciated. The main focus of this review is, therefore, one of mechanism and function, in addition to surveying the types of linkage observed and the methods employed for their identification. Case studies are presented for a variety of covalent flavoenzymes, from which general findings are beginning to emerge.
Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli
Authors: Muchmore CHRISTINE R. A. , Krahn Joseph M. , Smith Janet L. , Kim Jeong Hyun , Zalkin Howard 
Published: July 2010

AbstractCrystal structures of glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli have been determined to 2.0‐Å resolution in the absence of ligands, and to 2.5‐Å resolution with the feedback inhibitor AMP bound to the PRPP catalytic site. Glutamine PRPP amidotransferase (GPATase) employs separate catalytic domains to abstract nitrogen from the amide of glutamine and to transfer nitrogen to the acceptor substrate PRPP. The unliganded and AMP‐bound structures, which are essentially identical, are interpreted as the inhibited form of the enzyme because the two active sites are disconnected and the PRPP active site is solvent exposed. The structures were compared with a previously reported 3.0‐Å structure of the homologous Bacillus subtilis enzyme (Smith JL et al., 1994, Science 264:1427‐1433). The comparison indicates a pattern of conservation of peptide structures involved with catalysis and variability in enzyme regulatory functions. Control of glutaminase activity, communication between the active sites, and regulation by feedback inhibitors are addressed differently by E. coli and B. subtilis GPATases. The E. coli enzyme is a prototype for the metal‐free GPATases, whereas the B. subtilis enzyme represents the metal‐containing enzymes. The structure of the E. coli enzyme suggests that a common ancestor of the two enzyme subfamilies may have included an Fe‐S cluster.
Protein fold recognition without Boltzmann statistics or explicit physical basis
Authors: Huber Thomas , Torda Andrew E. 
Published: July 2010

AbstractWe present a fast method for finding optimal parameters for a low‐resolution (threading) force field intended to distinguish correct from incorrect folds for a given protein sequence. In contrast to other methods, the parameterization uses information from >107 misfolded structures as well as a set of native sequence‐structure pairs.In addition to testing the resulting force field's performance on the protein sequence threading problem, results are shown that characterize the number of parameters necessary for effective structure recognition.
Active‐site mobility in human immunodeficiency virus, type 1, protease as demonstrated by crystal structure of A28S mutant
Authors: Hong Lin , Hartsuck Jean A. , Foundling Steve , Ermoliefe Jacques , Tang Jordan 
Published: July 2010

AbstractThe mutation Ala28 to serine in human immunodeficiency virus, type 1, (HIV‐1) protease introduces putative hydrogen bonds to each active‐site carboxyl group. These hydrogen bonds are ubiquitous in pepsin‐like eukaryotic aspartic proteases. In order to understand the significance of this difference between HIV‐1 protease and homologous, eukaryotic aspartic proteases, we solved the three‐dimensional structure of A28S mutant HIV‐1 protease in complex with a peptidic inhibitor U‐89360E. The structure has been determined to 2.0 Å resolution with an R factor of 0.194. Comparison of the mutant enzyme structure with that of the wild‐type HIV‐1 protease bound to the same inhibitor (Hong L, Treharne A, Hartsuck JA, Foundling S, Tang J, 1996, Biochemistry 35:10627‐10633) revealed double occupancy for the Ser28 hydroxyl group, which forms a hydrogen bond either to one of the oxygen atoms of the active‐site carboxyl or to the carbonyl oxygen of Asp30. We also observed marked changes in orientation of the Asp25 catalytic carboxyl groups, presumably caused by the new hydrogen bonds. These observations suggest that catalytic aspartyl groups of HIV‐1 protease have significant conformational flexibility unseen in eukaryotic aspartic proteases. This difference may provide an explanation for some unique catalytic properties of HIV‐1 protease.
Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: Implications for redox potential regulation and flavodoxin folding
Authors: Steensma Elles , Nijman Melanie J. M. , Bollen Yves J. M. , Jager P. Adrie De , Van Den Berg Willy A. M. , Van Dongen Walter M.A. M. , Van Mierlo Carlo P. M. 
Published: July 2010

AbstractAs a first step to determine the folding pathway of a protein with an α/β doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five‐stranded parallel β‐sheet (β2‐βl‐β3‐β4‐β5) and five α‐helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 (kex < 10−5 s_1) and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous α/β doubly wound protein Che Y.
A folding pathway for βpep‐4 peptide 33mer: From unfolded monomers and β‐sheet sandwich dimers to well‐structured tetramers
Authors: Mayo Kevin H. , Ilyina Elea 
Published: July 2010

AbstractIt was recently reported that a de novo designed peptide 33mer, βpep‐4, can form well‐structured β‐sheet sandwich tetramers (Ilyina E, Roongta V, Mayo KH, 1997b, Biochemistry 56:5245‐5250). For insight into the pathway of βpep‐4 folding, the present study investigates the concentration dependence of βpep‐4 self‐association by using 1H‐NMR pulsedfield gradient (PFG)‐NMR diffusion measurements, and circular dichroism. Downfield chemically shifted αH resonances, found to arise only from the well‐structured βpep‐4 tetramer state, yield the fraction of tetramer within the oligomer equilibrium distribution. PFG‐NMR‐derived diffusion coefficients, D, provide a means for deriving the contribution of monomer and other oligomer states to this distribution. These data indicate that tetramer is the highest oligomer state formed, and that inclusion of monomer and dimer states in the oligomer distribution is sufficient to explain the concentration dependence of Dvalues for βpep‐4. Equilibrium constants calculated from these distributions [2.5 × 105 M−1 for M‐D and 1.2 × 104 M−1 for D‐T at 313 K] decrease only slightly, if at all, with decreasing temperature indicating a hydrophobically mediated, entropy‐driven association/folding process. Conformational analyses using NMR and CD provide a picture where “random coil” monomers associate to form molten globule‐like β‐sheet sandwich dimers that further associate and fold as well‐structured tetramers. βpep‐4 folding is thermodynamically linked to self‐association. As with folding of singlechain polypeptides, the final folding step to well‐structured tetramer βpep‐4 is rate limiting.