bluuebunny/crossref_metadata_2025 huggingface dataset:
Introduction of a [4Fe‐4S (S‐cys)4]+1,+2iron‐sulfur center into a four‐α helix protein using design parameters from the domain of the Fxcluster in the Photosystem I reaction center
Authors: Scott M. Paul , Biggins John
Published: July 2010

We describe the insertion of an iron‐sulfur center into a designed four α‐helix model protein. The model protein was re‐engineered by introducing four cysteine ligands required for the coordination of the mulinucleate cluster into positions in the main‐chain directly analogous to the domain predicted to ligand the interpeptide [4Fe‐4S (S‐cys)4] cluster, Fx, from PsaA and PsaB of the Photosystem I reaction center. This was achieved by inserting the sequence, CDGPGRGGTC, which is conserved in PsaA and PsaB, into interhelical loops 1 and 3 of the four α‐helix model. The holoprotein was characterized spectroscopically after insertion of the iron‐sulfur center in vitro. EPR spectra confirmed the cluster is a [4Fe‐4S] type, indicating that the cysteine thiolate ligands were positioned as designed. The midpoint potential of the iron‐sulfur center in the model holoprotein was determined via redox titration and shown to be −422 mV (pH 8.3,n= 1). The results support proposals advanced for the structure of the domain of the [4Fe‐4S] Fxcluster in Photosystem I based upon sequence predictions and molecular modeling. We suggest that the lower potential of the Fxcluster is most likely due to factors in the protein environment of Fxrather than the identity of the residues proximal to the coordinating ligands.
Intestinal fatty acid binding protein: A specific residue in one turn appears to stabilize the native structure and be responsible for slow refolding
Authors: Kim Keehyuk , Frieden Carl , Ramanathan Ragulan
Published: July 2010

The intestinal fatty acid binding protein is one of a class of proteins that are primarily β‐sheet and contain a large interior cavity into which ligands bind. A highly conserved region of the protein exists between two adjacent antiparallel strands (denoted as D and E in the structure) that are not within hydrogen bonding distance. A series of single, double, and triple mutations have been constructed in the turn between these two strands. In the wild‐type protein, this region has the sequence Leu 64/Gly 65/Val 66. Replacing Leu 64 with either Ala or Gly decreases the stability and the midpoint of the denaturation curve somewhat, whereas mutations at Gly 65 affect the stability slightly, but the protein folds at a rate similar to wild‐type and binds oleate. Val 66 appears not to play an important role in maintaining stability. All double or triple mutations that include mutation of Leu 64 result in a large and almost identical loss of stability from the wild‐type. As an example of the triple mutants, we investigated the properties of the Leu 64 Ser/Gly 65 Ala/Val 66 Asn mutant. As measured by the change in intrinsic fluorescence, this mutant (and similar triple mutants lacking leucine at position 64) folds much more rapidly than wild‐type. The mutant, and others that lack Leu 64, have far‐UV CD spectra similar to wild‐type, but a different near‐UV CD spectrum. The folded form of the protein binds oleate, although less tightly than wild‐type. Hydrogen/deuterium exchange studies using electrospray mass spectrometry indicate many more rapidly exchangeable amide protons in the Leu 64 Ser/Gly 65 Ala/Val 66 Asn mutant. We propose that there is a loss of defined structure in the region of the protein near the turn defined by the D and E strands and that the interaction of Leu 64 with other hydrophobic residues located nearby may be responsible for (1) the slow step in the refolding process and (2) the final stabilization of the structure. We suggest the possibility that this region of the protein may be involved in both an early and late step in refolding.
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

This 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

Human 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

A 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

A 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

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

The 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

Coliphage 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

Members 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

Cathepsin 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

The 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

Crystal 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

We 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

The 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

As 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

It 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.
Crystal structures of CheY from Thermotoga maritima do not support conventional explanations for the structural basis of enhanced thermostability
Authors: Usher Ken C. , De La Cruz Aida Flor A. , Dahlquist Fredrick W. , James Remington S. , Swanson Ronald V. , Simon Melvin I.
Published: July 2010

The crystal structure of CheY protein from Thermotoga maritime has been determined in four crystal forms with and without Mg++ bound, at up to 1.9 resolution. Structural comparisons with CheY from Escherichia coli shows substantial similarity in their folds, with some concerted changes propagating away from the active site that suggest how phosphorylated CheY, a signal transduction protein in bacterial chemotaxis, is recognized by its targets. A highly conserved segment of the protein (the “‐γ‐turn loop,” residues 55‐61), previously suggested to be a rigid recognition determinant, is for the first time seen in two alternative conformations in the different crystal structures. Although CheY from Thermotoga has much higher thermal stability than its mesophilic counterparts, comparison of structural features previously proposed to enhance thermostability such as hydrogen bonds, ion pairs, compactness, and hydrophobic surface burial would not suggest it to be so.
Formation and movement of Fe(III) in horse spleen, H‐ and L‐recombinant ferritins. A fluorescence study
Authors: Cavallo Stefano , Mei Giampiero , Stefanini Simonetta , Rosato Nicola , Finazzi‐Agró Alessandro , Chiancone Emilia
Published: July 2010

Iron oxidation and incorporation into apoferritins of different subunit composition, namely the recombinant H and L homopolymers and the natural horse spleen heteropolymer (10‐15% H), have been followed by steady‐state and time‐resolved fluorescence. After aerobic addition of 100 Fe(II) atoms/polymer, markedly different kinetic profiles are observed. In the rL‐homopolymer a slow monotonic fluorescence quenching is observed which reflects binding, slow oxidation at the threefold apoferritin channels, and diffusion into the protein cavity. In the rH‐homopolymer a fast fluorescence quenching is followed by a partial, slow recovery. The two processes have been attributed to Fe(II) binding and oxidation at the ferroxidase centers and to Fe(III) released into the cavity, respectively. The fluorescence kinetics of horse spleen apoferritin is dominated by the H chain contribution and resembles that of the H homopolymer. It brings out clearly that the rate of the overall process is limited by the rate at which Fe(III) leaves the ferroxidase centers of the H chains where binding of incoming Fe(II) and its oxidation take place. The data obtained upon stepwise addition of iron and the results of optical absorption measurements confirm this picture. The correspondence between steady‐state and time‐resolved data is remarkably good; this is manifest when the latter are used to calculate the change in fluorescence intensity as apparent in the steady‐state measurements.
Comprehensive assessment of automatic structural alignment against a manual standard, the scop classification of proteins
Authors: Gerstein Mark , Levitt Michael
Published: July 2010

We apply a simple method for aligning protein sequences on the basis of a 3D structure, on a large scale, to the proteins in the scop classification of fold families. This allows us to assess, understand, and improve our automatic method against an objective, manually derived standard, a type of comprehensive evaluation that has not yet been possible for other structural alignment algorithms. Our basic approach directly matches the backbones of two structures, using repeated cycles of dynamic programming and least‐squares fitting to determine an alignment minimizing coordinate difference. Because of simplicity, our method can be readily modified to take into account additional features of protein structure such as the orientation of side chains or the location‐dependent cost of opening a gap. Our basic method, augmented by such modifications, can find reasonable alignments for all but 1.5% of the known structural similarities in scop, i.e., all but 32 of the 2,107 superfamily pairs. We discuss the specific protein structural features that make these 32 pairs so difficult to align and show how our procedure effectively partitions the relationships in scop into different categories, depending on what aspects of protein structure are involved (e.g., depending on whether or not consideration of side‐chain orientation is necessary for proper alignment). We also show how our pairwise alignment procedure can be extended to generate a multiple alignment for a group of related structures. We have compared these alignments in detail with corresponding manual ones culled from the literature. We find good agreement (to within 95% for the core regions), and detailed comparison highlights how particular protein structural features (such as certain strands) are problematical to align, giving somewhat ambiguous results. With these improvements and systematic tests, our procedure should be useful for the development of scop and the future classification of protein folds. Supplementary material is available at http://bioinfo.mbb.yale.edu/align.
Purification and crystallization of the CDK‐associated protein phosphatase KAP expressed in escherichia coli
Authors: Hanlon Neil , Barford David
Published: July 2010

The kinase associated phosphatase (KAP) is a human dual specificity protein phosphatase that dephosphorylates the cell cycle control protein, cyclin dependent kinase‐2 on Thr 160 in a cyclin dependent manner (Poon & Hunter, 1995). We report here the over‐expression of KAP in Escherichia coli as an N‐terminal His‐tagged protein using a modified pET‐28a T7‐expression vector. The recombinant protein was purified to homogeneity and crystallized. The crystals diffract to 2.3 Ǎ resolution when exposed to synchrotron radiation and belong to space group P6122, or its enantiomorph P6522, with unit cell dimensions a= b 74.5 Ǎ, c = 139.5 Ǎ.
Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: A 2.2 Å resolution structure in a hexagonal crystal form
Authors: Yan Youwei , Li Ying , Munshi Sanjeev , Sardana Vinod , Cole James L. , Sardana Mohinder , Kuo Lawarence C. , Chen Zhongguo , Steinkuehler Christian , Tomei Licia , Francesco Raffaele De
Published: July 2010

The crystal structure of the NS3 protease of the hepatitis C virus (BK strain) has been determined in the space group P6322 to a resolution of 2.2 Å. This protease is bound with a 14‐mer peptide representing the central region of the NS4A protein. There are two molecules of the NS31_180‐NS4A21′‐34′ complex per asymmetric unit. Each displays a familiar chymotrypsin‐like fold that includes two β‐barrel domains and four short α‐helices. The catalytic triad (Ser‐139, His‐57, and Asp‐81) is located in the crevice between the β‐barrel domains. The NS4A peptide forms an almost completely enclosed peptide surface association with the protease. In contrast to the reported H strain complex of NS3 protease‐NS4A peptide in a trigonal crystal form (Kim JL et al., 1996, Cell 87:343‐355), the N‐terminus of the NS3 protease is well‐ordered in both molecules in the asymmetric unit of our hexagonal crystal form. The folding of the N‐terminal region of the NS3 protease is due to the formation of a three‐helix bundle as a result of crystal packing. When compared with the unbound structure (Love RA et al., 1996, Cell 87:331‐342), the binding of the NS4A peptide leads to the ordering of the N‐terminal 28 residues of the NS3 protease into a β‐strand and an α‐helix and also causes local rearrangements important for a catalytically favorable conformation at the active site. Our analysis provides experimental support for the proposal that binding of an NS4A‐mimicking peptide, which increases catalytic rates, is necessary but not sufficient for formation of a well‐ordered, compact and, hence, highly active protease molecule.
Heterogeneity of packing: Structural approach
Authors: Kurochkina Natalya , Privalov George
Published: July 2010

Analysis of the heterogeneity of packing in proteins showed that different groups of the protein preferentially contribute to low‐ or high‐density regions. Statistical distribution reveals the two preferable values for packing density in the form of two peaks. One peak occurs in the range of densities 0.55‐0.65, the other occurs in the range 0.75‐0.8. The high‐density peak is originated primarily by high packing inside the hydrogen bonded backbone and to some extent by side chains. Polar/charged and apolar side chains both contribute to the low‐density peak. The average packing density values of individual atomic groups significantly vary for backbone atoms as well as for side chain atoms. The carbonyl oxygen atoms of protein backbone and the end groups of side chains show lower packing density than the rest of the protein. The side‐chain atomic groups of a secondary structure element when packed against the neighboring secondary structure element form stronger contacts with the side chains of this element than with its backbone. Analysis of the low‐density regions around each buried peptide group was done for the set of proteins with different types of packing, including α‐α, α‐β, and β‐β packing. It was shown that cavities are regularly situated in the groove of secondary structure element packed against neighboring elements for all types of packing. Low density in the regions surrounding the peptide groups and the end groups of side chains can be explained by their positioning next to a cavity formed upon the association of secondary structure elements. The model proposed can be applied to the analysis of protein internal motions, mechanisms of cellular signal transduction, diffusion through protein matrix, and other events.
A cognate tRNA specific conformational change in glutaminyl‐tRNA synthetase and its implication for specificity
Authors: Mandal Amit Kumar , Bhattacharyya Anusree , Bhattacharyya Subhra , Bhattacharyya Tista , Roy Siddhartha
Published: July 2010

Conformational changes that occur upon substrate binding are known to play crucial roles in the recognition and specific aminoacylation of cognate tRNA by glutaminyl‐tRNA synthetase. In a previous study we had shown that glutaminyl‐tRNA synthetase labeled selectively in a nonessential sulfhydryl residue by an environment sensitive probe, acrylodan, monitors many of the conformational changes that occur upon substrate binding. In this article we have shown that the conformational change that occurs upon tRNAGln binding to glnRS/ATP complex is absent in a noncognate tRNA tRNAGlu‐glnRS/ATP complex. CD spectroscopy indicates that this cognate tRNAGln‐induced conformational change may involve only a small change in secondary structure. The Van't Hoff plot of cognate and noncognate tRNA binding in the presence of ATP is similar, suggesting similar modes of interaction. It was concluded that the cognate tRNA induces a local conformational change in the synthetase that may be one of the critical elements that causes enhanced aminoacylation of the cognate tRNA over the noncognate ones.
Crystallization and preliminary X‐ray analysis of a 1:1 complex between a designed monomeric interferon‐gamma and its soluble receptor
Authors: Randal M. , Kossiakoff A. A.
Published: July 2010

A variant of human interferon‐gamma (IFN‐γ) has been created in which the two chains of the homodimeric cytokine were linked N‐ to C‐terminus by an eight residue polypeptide linker. The sequence of this linker was derived from a loop in bira bifunctional protein, and was determined from a structural database search. This “single‐chain” variant was used to create an IFN‐γ molecule that binds only a single copy of the α‐chain receptor, rather than the 2 α‐chain receptor: 1 IFN‐γ binding stoichiometry observed for the native hormone. Crystals have been grown of a 1:1 complex between this single‐chain molecule and the extracellular domain of its α‐chain receptor. These crystals diffract beyond 2.0 Å, significantly better than the 2.9 Å observed for the native 2:1 complex. Density calculations suggest these crystals contain two complexes in the asymmetric unit; a self‐rotation function confirms this conclusion.
Incidental adenocarcinoma of the prostate: The role of repeat transurethral resection in staging
Authors: Paul Sonda L. , Barton Grossman H. , MacGregor Robert J , Gikas Paul W.
Published: July 2010

Accurate staging of patients with incidental prostatic adenocarcinoma (A1 vs A2) is crucial to the selection of appropriate treatment. To evaluate the potential sampling error in specimens obtained by transurethral resection, repeat resection was performed on 31 patients pathologically staged as A1 (five or less chips with tumor). Second specimens showed no tumor in 22 (71 %), stage A1 in six (20%), and stage A2 in three (9%). The weight of tissue removed at reresection was greater in patients found to have more extensive involvement (P < 0.005). No patient with initial Gleason score 2, 3, or 4 had stage A2 at reresection. Repeat resection is not routinely necessary, but may be helpful when the tumor is high grade but of minimal extent, in an anxious younger patient who may wish therapy if disease persists, or where some suspicion of an incomplete resection exists.
Susceptibility of the prostate cancer cell to different physical, hormonal, and chemical agents: Present status and theoretical prospects for improved prostate cancer therapy
Authors: Hechter Oscar
Published: July 2010

The benefits and limitations of present modes of treatment of prostatic cancer with physical, hormonal, and chemical agents are briefly reviewed. The theoretical possibility that the heterogenous clones that comprise prostatic cancer might be killed or controlled by selective targeting of cytotoxic agents (actual or potential) to malignant cells is discussed in terms of two types of delivery vehicles to provide for cell selectivity: (1) monoclonal antibodies against specific cell surface markers on cancer cells and (2) steroid ligands for receptors present in clones of malignant cells. The problems and prospects of these selective delivery systems as potential therapeutic modalities for prostatic cancer are considered.
Hyperthermia in the treatment of prostate cancer
Authors: Servadio C. , Leib Z.
Published: July 2010

Hyperthermia—the sustained heating of tissues to temperatures of 42oC to 43.5oC− increasingly is being studied by scientists as a new tool to destroy cancer cells, either alone or in conjunction with other well established forms of treatment, such as radiotherapy and chemotherapy. Many studies on tissue cultures, on animals and also on humans, have established the fact that this new nonconventional adjuvant form of treatment is actually effective. Progress is being made in the development of the necessary technology for delivering the desired and controlled form of heat to the diseased site, while protecting the surrounding healthy tissues from irreversible damage. Initial clinical trials in this direction are very promising and seem to offer new avenues in the methods of treatment for cancer of the prostate.
Immunotherapy of prostate cancer: A review
Authors: Guinan P. , Ray P. , Shaw M.
Published: July 2010

Tumors are antigenic and patients can mount an immune response against their tumors. Prostate cancer patients are sensitized to their malignancies and immunotherapeutic efforts have been successful. As with other tumors, it is unlikely that immunotherapy alone will be curative, but will play a role in combination therapy along with surgery, radiation, and chemotherapy. But before its rightful place can be delineated, additional clinical and laboratory studies must be completed.
Laser ablation of AgSbS2 and cluster analysis by time‐of‐flight mass spectrometry
Authors: Houška Jan , Peña‐Méndez Eladia Maria , Kolář Jakub , Frumar Miloslav , Wágner Tomáš , Havel Josef
Published: July 2010

Thin films of AgSbS2 are important for phase‐change memory applications. This solid is deposited by various techniques, such as metal organic chemical vapour deposition or laser ablation deposition, and the structure of AgSbS2(s), as either amorphous or crystalline, is already well characterized. The pulsed laser ablation deposition (PLD) of solid AgSbS2 is also used as a manufacturing process. However, the processes in plasma have not been well studied. We have studied the laser ablation of synthesized AgSbS2(s) using a nitrogen laser of 337 nm and the clusters formed in the laser plume were identified. The ablation leads to the formation of various single charged ternary AgpSbqSr clusters. Negatively charged AgSbS, AgSb2S, AgSb2S, AgSb2S and positively charged ternary AgSbS+, AgSb2S+, AgSb2S, AgSb2S clusters were identified. The formation of several singly charged Ag+, Ag, Ag, Sb, Sb, S ions and binary AgpSr clusters such as AgSb, Ag3S−, SbS (r = 1–5), Sb2S−, Sb2S, Sb3S (r = 1–4) and AgS, SbS+, SbS, Sb2S+, Sb2S, Sb3S (r = 1–4), AgSb was also observed. The stoichiometry of the clusters was determined via isotopic envelope analysis and computer modeling. The relation of the composition of the clusters to the crystal structure of AgSbS2 is discussed. Copyright © 2009 John Wiley & Sons, Ltd.
Brownfields—A Canadian Perspective
Authors: Velderman B. J. , Zapf‐Gilje R. , Fortin F. , DuBois D.
Published: July 2010

Barriers to redevelopment of contaminated lands have led to vacant or underutilized sites termed “brown fields.” These barriers fall into six categories: regulatory, technical/scientific, legal/liability, financial, urbanplanning, and communications. The lack of protection to innocent parties, such as developers and lenders, and the lack of guidance for applying sitespecific and risk‐based remediation approaches are two key barriers to redevelopment. This article presents guiding principles for redeveloping brownfields and recommends best practices toward overcoming existing barriers to such redevelopment. Success stories of redeveloped contaminated industrial sites are provided to illuminate the effectiveness of the best practices approach.
Recent Developments in Cleanup Technologies
Authors: Simon John A.
Published: July 2010

Although the reaction mechanics are somewhat mysterious, the use of iron forin situgroundwater treatment has recently gained considerable attention and respect in the remediation industry. The basic scientific principles of both applications of iron have been known for over a century; however, both were nearly unheard of as remediation technologies five years ago. Both technologies have a strong potential for widespread use. They are commercially available, have been proven in field studies, are less expensive than traditional pump and treat technologies, and, in many types of groundwater systems, may be able to meet difficult‐to‐achieve groundwater treatment standards. As these technologies continue to undergo development, there could be considerably more aggressive applications used to treat ground‐water containing high concentrations of chlorinated organics and DNAPLs.
Time domain constrained H∞‐synthesis
Authors: Apkarian Pierre , Ravanbod‐Hosseini Laleh , Noll Dominikus
Published: July 2010

Structured H∞‐synthesis subject to time domain performance specifications is introduced. Our method allows to control trajectories of the linearized system and the underlying nonlinear dynamics simultaneously. A non‐smooth bundle optimization method for this class of programs is proposed and discussed. Our approach is tested against two benchmark studies: control of a rotational actuator to attenuate vibration noise, and control of a continuous crystallizer. Our algorithm gives a local convergence certificate and is suited for systems with large state dimension. Copyright © 2010 John Wiley & Sons, Ltd.
Inequalities in probability theory and turán‐type problems for graphs with colored vertices
Authors: Sidorenko A. F.
Published: July 2010

The “best” inequalities of type P{(ζ, η)⊂ E} ≧f(P{η⊂ D1}P{η⊂Dm}) for independent and identically distributed random elements ζ and η can be reduced to Turán‐type problems for graphs with colored vertices. In the present work we describe a finite algorithm for obtaining the asymptotical solution for an arbitrary problem of such type. In the case of two colors we obtain the final form of asymptotic solution without using the algorithm.