bluuebunny/crossref_metadata_2025_split huggingface dataset:
The pH dependence of hydrogen‐deuterium exchange in trp repressor: The exchange rate of amide protons in proteins reflects tertiary interactions, not only secondary structure
Authors: Finucane Michael D. , Jardetzky Oleg 
Published: July 2010

AbstractThe pH dependence of amide proton exchange rates have been measured for trp‐repressor. One class of protons exchanges too fast to be measured in these experiments. Among the protons that have measurable hydrogen‐deuterium exchange rates, two additional classes may be distinguished. The second class of protons are in elements of secondary structure that are mostly on the surface of the protein, and exchange linearly with increasing base concentration (log kex versus pH). The third class of amide protons is characterized by much higher protection against exchange at higher pH. These protons are located in the core of the protein, in helices B and C. The exchange rate in the core region does not increase linearly with pH, but rather goes through a minimum around pH 6.The mechanism of exchange for the slowly exchanging core protons is interpreted in terms of the two‐process model of Hilton and Woodward (1979, Biochemistry 75:5834–5841), i.e., exchange through both a local mechanism that does not require unfolding of the protein, and a mechanism involving global unfolding of the protein. The increase in exchange rates at low pH is attributed to a partial unfolding of the repressor. It is concluded that the formation of secondary structure alone is insufficient to account for the high protection factors seen in the core of native proteins at higher pH, and that tertiary interactions are essential to stabilize the structure.
Crystal structure of cod liver class I alcohol dehydrogenase: Substrate pocket and structurally variable segments
Authors: Ramaswamy S. , Ahmad Mustapha El , Danielsson Olle , Jörnvall Hans , Eklund Hans 
Published: July 2010

AbstractThe structural framework of cod liver alcohol dehydrogenase is similar to that of horse and human alcohol dehydrogenases. In contrast, the substrate pocket differs significantly, and main differences are located in three loops. Nevertheless, the substrate pocket is hydrophobic like that of the mammalian class I enzymes and has a similar topography in spite of many main‐chain and side‐chain differences. The structural framework of alcohol dehydrogenase is also present in a number of related enzymes like glucose dehydrogenase and quinone oxidoreductase. These enzymes have completely different substrate specificity, but also for these enzymes, the corresponding loops of the substrate pocket have significantly different structures. The domains of the two subunits in the crystals of the cod enzyme further differ by a rotation of the catalytic domains by about 6°. In one subunit, they close around the coenzyme similarly as in coenzyme complexes of the horse enzyme, but form a more open cleft in the other subunit, similar to the situation in coenzyme‐free structures of the horse enzyme. The proton relay system differs from the mammalian class I alcohol dehydrogenases. His 51, which has been implicated in mammalian enzymes to be important for proton transfer from the buried active site to the surface is not present in the cod enzyme. A tyrosine in the corresponding position is turned into the substrate pocket and a water molecule occupies the same position in space as the His side chain, forming a shorter proton relay system.
Roles of Glu 349 and Asp 352 in membrane insertion and translocation by diphtheria toxin
Authors: Kaul Poonam , Silverman Jared , Shen Wei Hai , Blanke Steven R. , Huynh Paul D. , Finkelstein Alan , John Collier R. 
Published: July 2010

AbstractAcidic conditions within the endosomal lumen induce the T domain of receptor‐bound diphtheria toxin (DT) to insert into the endosomal membrane and mediate translocation of the toxin's catalytic domain to the cytosol. A conformational rearrangement in the toxin occurring near pH 5 allows a buried apolar helical hairpin of the native T domain (helices TH8 and TH9) to undergo membrane insertion. If the inserted hairpin spans the bilayer, as hypothesized, then the two acidic residues within the TL5 interhelical loop, Glu 349 and Asp 352, should become exposed at the neutral cytosolic face of the membrane and reionize. To investigate the roles of these residues in toxin action, we characterized mutant toxins in which one or both acidic residues had been replaced with nonionizable ones. Each of two double mutants examined showed a several‐fold reduction in cytotoxicity in 24‐h Vero cell assays (sixfold for E349A+D352A and fourfold for E349Q+D352N), whereas the individual E349Q and D352N mutations caused smaller reductions in toxicity. The single and double mutations also attenuated the toxin's ability to permeabilize Vero cells to Rb+ at low pH and decreased channel formation by the toxin in artificial planar bilayers. Neither of the double mutations affected the pH‐dependence profile of the toxin's conformational rearrangement in solution, as measured by binding of the hydrophobic fluorophore, 2‐p‐toluidinyl‐naphthalene 6‐sulfonate. The results demonstrate that, although there is no absolute requirement for an acidic residue within the TL5 loop for toxicity, Glu 349 and Asp 352 do significantly enhance the biological activity of the protein. The data are consistent with a model in which ionization of these residues at the cytosolic face of the endosomal membrane stabilizes the TH8/TH9 hairpin in a transmembrane configuration, thereby facilitating channel formation and translocation of the toxin's catalytic chain.
Xylanase homology modeling using the inverse protein folding approach
Authors: Chen Xiannong , Whitmire David , Bowen J. Phillip 
Published: July 2010

AbstractXylanase has been used in wood pulp bleaching in an effort to reduce chlorine release into the environment and pollution associated with paper production. The three‐dimensional structure of xylanase is important to enable better understanding of the enzyme mechanism and to help design a more thermostable xylanase mutant. At the time this work was begun, there was no sequence homologous protein available for traditional sequence‐based homology modeling. In order to circumvent this problem, the inverse protein folding approach was undertaken to find a suitable template structure. Model structures of Bacillus circulans xylanase were built based on the database search results of related proteins. The model structures were refined and compared to the recently solved xylanase X‐ray crystal structure. The overall structural similarity between the theoretical model and experimental structure demonstrate the usefulness of this approach. Disagreement in folding topology, however, warrants further research into the inverse protein folding approach.
Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: Characterization of the active site and evidence for an interdomain carboxy‐terminal helix in ornithine transcarbamoylase
Authors: Murata Lauren B. , Schachman H.K. 
Published: July 2010

AbstractPredictions of tertiary structures of proteins from their amino acid sequences are facilitated greatly when the structures of homologous proteins are known. On this basis, structural features of Escherichia coli ornithine transcarbamoylase (OTCase) were investigated by site‐directed mutagenesis experiments based on the known tertiary structure of the catalytic (c) chain of E. coli aspartate transcarbamoylase (ATCase). In ATCase, each c chain is composed of two globular domains connected by two interdomain helices, one of which is near the C‐terminus and is critical for the in vivo folding of the chains and their assembly into trimers. Each active site is located at the interface between two chains and requires the participation of residues from each of the adjacent chains. OTCase, a trimeric enzyme, has been proposed to be similar in structure to the ATCase trimer on the basis of sequence identity (32%), the nature of the reaction catalyzed by the enzyme, and secondary structure predictions. As shown here, analysis of OTCase and ATCase sequences revealed extensive evolutionary conservation in portions corresponding to the ATCase active site and the C‐terminal helix. Truncations and substitutions within the predicted C‐terminal helix of OTCase had effects on activity and thermal stability strikingly similar to those caused by analogous alterations in ATCase. Similarly, substitutions at either of two conserved residues, Ser 55 and Lys 86, in the proposed active site of OTCase had deleterious effects parallel to those caused by the analogous ATCase substitutions. Hybrid trimers comprised of chains from both these relatively inactive OTCase mutants exhibited dramatically increased activity, as predicted for shared active sites located at the chain interfaces. These results strongly support the hypothesis that the tertiary and quaternary structures of the two enzymes are similar.
Subunit dissociations in natural and recombinant hemoglobins
Authors: Manning Lois R. , Jenkins W. Terry , Hess John R. , Vandegriff Kim , Winslow Robert M. , Manning James M. 
Published: July 2010

AbstractA precise and rapid procedure employing gel filtration on Superose‐12 to measure the tetramer‐dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val‐6(β) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the α1β2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.
Comparison of the structures of the cyclotheonamide a complexes of human α‐thrombin and bovine β‐trypsin
Authors: Ganesh V. , Tulinsky A. , Lee A.Y. , Clardy J. 
Published: July 2010

AbstractThrombin, a trypsin‐like serine protease present in blood, plays a central role in the regulation of thrombosis and hemostasis. A cyclic pentapeptide, cyclotheonamide A (CtA), isolated from sponges of the genus Theonella, inhibits thrombin, trypsin, and certain other serine proteases. Enzyme inhibition data for CtA indicate that it is a moderate inhibitor of α‐thrombin (Ki = 1.0 nM), but substantially more potent toward trypsin (Ki = 0.2 nM). The comparative study of the crystal structures of the CtA complexes of α‐thrombin and β‐trypsin reported here focuses on structure‐function relationships in general and the enhanced specificity of trypsin, in particular.The crystal structures of the CtA complexes of thrombin and trypsin were solved and refined at 1.7 and 2.0 Å resolution, respectively. The structures show that CtA occupies the active site with the Pro‐Arg motif positioned in the S2 and S1 binding sites. The α‐keto group of CtA is involved in a tetrahedral intermediate hemiketal structure with Ser 195 OG of the catalytic triad and is positioned within bonding distance from, and orthogonal to, the re‐face of the carbonyl of the arginine of CtA. As in other productive binding modes of serine proteases, the Ser 214‐Gly 216 segment runs in a twisted antiparallel β‐strand manner with respect to the diaminopropionic acid (Dpr)‐Arg segment of CtA. The Tyr 60A‐Thr 601 insertion loop of thrombin makes a weak aromatic stacking interaction with the v‐Tyr of CtA through Trp 60D. The Glu 39 Tyr and Leu 41 Phe substitutions in trypsin produce an enhanced aromatic interaction with D‐Phe of CtA, which also leads to different orientations of the side chains of D‐Phe and the V‐Tyr.The comparison of the CtA complexes of thrombin and trypsin shows that the gross structural features of both in the active site region are the same, whereas the differences observed are mainly due to minor insertions and substitutions. In trypsin, the substitution of Ile 174‐Arg 175 by Gly 174‐Gln 175 makes the S3 aryl site more polar because the Arg 175 side chain is directed away from thrombin and into the solvent, whereas Gln 175 is not. Because the site is occupied by the Dpr group of CtA, the occupancy of the S3 site is better in trypsin than in thrombin. In trypsin, the D‐Phe side chain of CtA fits between Tyr 39 and Phe 41 in a favorable manner, whereas in thrombin, these residues are Glu 39 and Leu 41. The higher degree of specificity for trypsin is most likely the result of these substitutions and the absence of the fairly rigid Tyr 60A‐Thr 601 insertion loop of thrombin, which narrows access to the active site and forces less favorable orientations for the D‐Phe and v‐Tyr residues.
Structural investigation of the alpha‐1‐antichymotrypsin: Prostate‐specific antigen complex by comparative model building
Authors: Villoutreix Bruno O. , Teleman Olle , Lilja Hans , Pettersson Kim , Lövgren Timo 
Published: July 2010

AbstractProstate‐specific antigen (PSA), produced by prostate cells, provides an excellent serum marker for prostate cancer. It belongs to the human kallikrein family of enzymes, a second prostate‐derived member of which is human glandular kallikrein‐1 (hK2). Active PSA and hK2 are both 237‐residue kallikrein‐like proteases, based on sequence homology. An hK2 model structure based on the serine protease fold is presented and compared to PSA and six other serine proteases in order to analyze in depth the role of the surface‐accessible loops surrounding the active site. The results show that PSA and hK2 share extensive structural similarity and that most amino acid replacements are centered on the loops surrounding the active site. Furthermore, the electrostatic potential surfaces are very similar for PSA and hK2.PSA interacts with at least two serine protease inhibitors (serpins): alpha‐1‐antichymotrypsin (ACT) and protein C inhibitor (PCI). Three‐dimensional model structures of the uncleaved ACT molecule were developed based upon the recent X‐ray structure of uncleaved antithrombin. The serpin was docked both to PSA and hK2. Amino acid replacements and electrostatic complementarities indicate that the overall orientation of the proteins in these complexes is reasonable. In order to investigate PSA's heparin interaction sites, electrostatic computations were carried out on PSA, hK2, protein C, ACT, and PCI. Two heparin binding sites are suggested on the PSA surface and could explain the enhanced complex formation between PSA and PCI, while inhibiting the formation of the ACT‐PSA complex. PSA, hK2, and their preliminary complexes with ACT should facilitate the understanding and prediction of structural and functional properties for these important proteins also with respect to prostate diseases.
Purification and characterization of dihydroorotate dehydrogenase a from lactococcus lactis, crystallization and preliminary X‐ray diffraction studies of the enzyme
Authors: Nielsen Finn S. , Jensen Kaj Frank , Rowland Paul , Larsen Sine 
Published: July 2010

AbstractLactococcus lactis is the only organism known to contain two dihydroorotate dehydrogenases, i.e., the A‐ and B‐forms. In this paper, we report the overproduction, purification, and crystallization of dihydroorotate dehydrogenase A. In solution, the enzyme is bright yellow. It is a dimer of subunits (34 kDa) that contain one molecule of flavin mononucleotide each. The enzyme shows optimal function in the pH range 7.5–9.0. It is specific for L‐dihydroorotate as substrate and can use dichlorophenolindophenol, potassium hexacyanoferrate(III), and, to a lower extent, also molecular oxygen as acceptors of the reducing equivalents, whereas the pyridine nucleotide coenzymes (NAD+, NADP+) and the respiratory quinones (i.e., vitamins Q6, Q10 and K2) were inactive. The enzyme has been crystallized from solutions of 30% polyethylene glycol, 0.2 M sodium acetate, and 0.1 M Tris‐HC1, pH 8.5. The resulting yellow crystals diffracted well and showed little sign of radiation damage during diffraction experiments. The crystals are monoclinic, space group P2, with unit cell dimensions a = 54.19 Å, b = 109.23 Å, c = 67.17 Å, and β = 104.5°. A native data set has been collected with a completeness of 99.3% to 2.0 Å and an Rsym value of 5.2%. Analysis of the solvent content and the self‐rotation function indicates that the two subunits in the asymmetric unit are related by a noncrystallographic twofold axis perpendicular to the crystallographic b and c axes.
Protein design automation
Authors: Dahiyat Bassil I. , Mayo Stephen L. 
Published: July 2010

AbstractWe have conceived and implemented a cyclical protein design strategy that couples theory, computation, and experimental testing. The combinatorially large number of possible sequences and the incomplete understanding of the factors that control protein structure are the primary obstacles in protein design. Our protein design automation algorithm objectively predicts protein sequences likely to achieve a desired fold. Using a rotamer description of the side chains, we implemented a fast discrete search algorithm based on the Dead‐End Elimination Theorem to rapidly find the globally optimal sequence in its optimal geometry from the vast number of possible solutions. Rotamer sequences were scored for steric complementarity using a van der Waals potential. A Monte Carlo search was then executed, starting at the optimal sequence, in order to find other high‐scoring sequences. As a test of the design methodology, high‐scoring sequences were found for the buried hydrophobic residues of a homodimeric coiled coil based on GCN4‐p1. The corresponding peptides were synthesized and characterized by CD spectroscopy and size‐exclusion chromatography. All peptides were dimeric and nearly 100% helical at 1 °C, with melting temperatures ranging from 24 °C to 57 °C. A quantitative structure activity relation analysis was performed on the designed peptides, and a significant correlation was found with surface area burial. Incorporation of a buried surface area potential in the scoring of sequences greatly improved the correlation between predicted and measured stabilities and demonstrated experimental feedback in a complete design cycle.
A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: Identification of duplicated repeats and potential active site residues
Authors: Ponting C.P. , Kerr I.D. 
Published: July 2010

AbstractPhosphatidylcholine‐specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols. Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein. Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities both to each other, and to two other classes of phospholipid‐specific enzymes, bacterial cardiolipin synthases, and eukaryotic and bacterial phosphatidylserine synthases, indicating that these enzymes form an homologous family. This family is suggested also to include two Poxviridae proteins of unknown function (p37K and protein K4), a bacterial endonuclease (nuc), an Escherichia coli putative protein (o338) containing an N‐terminal domain showing similarities with helicase motifs V and VI, and a Synechocystis sp. putative protein with a C‐terminal domain likely to possess a DNA‐binding function. Surprisingly, four regions of sequence similarity that occur once in nuc and o338, appear twice in all other homologues, indicating that the latter molecules are bi‐lobed, having evolved from an ancestor or ancestors that underwent a gene duplication and fusion event. It is suggested that, for each of these enzymes, conserved histidine, lysine, aspartic acid, and/or asparagine residues may be involved in a two‐step ping‐pong mechanism involving an enzyme‐substrate intermediate.
Preliminary crystallographic studies of an anti‐HIV‐1 protease antibody that inhibits enzyme activity
Authors: Lescar Julien , Stouracova Renata , Riottot Marie‐Madeleine , Chitarra Véronique , Brynda Jiri , Fabry Milan , Horejsi Magda , Sedlacek Juraj , Bentley Graham A. 
Published: July 2010

AbstractF11.2.32, a monoclonal antibody directed against the HIV‐1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme. The antibody cross‐reacts with peptides 36–46 and 36–57 from the protease. Crystals of the Fab have been obtained both in the free state and as complexes formed with the protease peptide fragments, 36–46 and 36–57. Diffraction data have been collected for the free and complexed forms of Fab F11.2.32 and preliminary models for the crystal structures were obtained by molecular replacement.
Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S‐transferase, 1–1
Authors: Wang Jibo , Barycki Joseph J. , Colman Roberta F. 
Published: July 2010

AbstractReaction of rat liver glutathione S‐transferase, isozyme 1–1, with 4‐(fluorosulfonyl)benzoic acid (4‐FSB), a xenobiotic substrate analogue, results in a time‐dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with l‐chloro‐2,4‐dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4‐FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min−1. S‐Hexylglutathione or the xenobiotic substrate analogue, 2,4‐dinitrophenol, protects against inactivation of the enzyme by 4‐FSB, whereas S‐methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5‐3H]‐4‐FSB into the enzyme in the absence and presence of S‐hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4‐FSB, but only Tyr 8 is protected against 4‐FSB by S‐hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4‐FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4‐FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme‐catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme‐bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the ‐SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S‐transferase, but is not required for enzyme activity.