bluuebunny/crossref_metadata_2025_split huggingface dataset:
The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc‐dependent binding to an anionic surface
Authors: Dela Raul A. Cadena , Colman Robert W. 
Published: July 2010

AbstractThe histidine‐glycine‐rich region of the light chain of cleaved high molecular weight kininogen (HK) is thought to be responsible for binding to negatively charged surfaces and initiation of the intrinsic coagulation, fibrinolytic, and kinin‐forming systems. However, the specifically required amino acid sequences have not been delineated. An IgG fraction of a monoclonal antibody (MAb) C11C1 to the HK light chain was shown to inhibit by 66% the coagulant activity and by 57% the binding of HK to the anionic surface of kaolin at a concentration of 1.5 μM and 27 μM, respectively. Proteolytic fragments of HK were produced by successive digestion with human plasma kallikrein and factor XIa (FXIa). Those polypeptides that bound tightly (Kd = 0.77 nM) to a C11C1 affinity column were eluted at pH 3.0 and purified by membrane filtration. On 15% SDS polyacrylamide electrophoresis, the approximate M was 7.3 kDa (range 6.2–8.1 kDa). Based on N‐terminal sequencing, this polypeptide (l2), which extends from the histidine residue 459 to a lysine at position 505, 509, 511, 512, 515, or 520, inhibits by 50% the coagulant activity expressed by HK at a concentration of 22 μM. The synthetic peptide HGLGHGH representing the N‐terminal of the l2 fragment was synthesized, tested, and found at 4 mM to inhibit the procoagulant activity of HK 50%. A synthetic heptadecapeptide, HGLGHGHEQQHGLGHGH (residues 459–475) included within the l2 fragment, and with the ability to bind zinc, inhibited 50% of the HK coagulant activity at a concentration of 325 μM in the absence and presence of added Zn2+ (30 μM). The specific binding of 125I‐HK to a negatively charged surface (kaolin) was inhibited 50% by unlabeled HK (5 μM). HGLGHGH, at a concentration of 7.0 mM, inhibited the binding to kaolin by 50%. The heptadecapeptide inhibited the specific binding of 125I‐HK to kaolin by 50%, at a concentration of 2.3 mM, in the absence of Zn2+. In contrast, when Zn2+ was added, the concentration to achieve 50% inhibition decreased to 630 μM, indicating that Zn2+ was required to attain a favorable conformation for binding. Moreover, the l2 fragment was found to inhibit 50% of the 125I‐HK binding to kaolin at a concentration of 380 μM. These results suggest that residues contained within the l2 fragment, notably HGLGHGHEQQHGLGHGH, serves as a primary structural feature for binding to a negatively charged surface.
A series of point mutations reveal interactions between the calcium‐binding sites of calmodulin
Authors: Starovasnik Melissa A. , Klevit Rachel E. , Su Dai‐Rong , Beckingham Kathy 
Published: July 2010

AbstractCalmodulin is a member of the “EF‐hand” family of Ca2+‐binding proteins. It consists of two homologous globular domains, each containing two helix‐loop‐helix Ca2+‐binding sites. To examine the contribution of individual Ca2+‐binding sites to the Ca2+‐binding properties of CaM, a series of four site‐directed mutants has been studied. In each, the glutamic acid at position 12 in one of the four Ca2+‐binding loops has been changed to a glutamine. One‐dimensional 1H‐NMR has been used to monitor Ca2+‐induced changes in the mutant proteins, and the spectral changes observed for each mutant have been compared to those for wild‐type CaM. In this way, the effect of each mutation on both the mutated site and the other Ca2+‐binding sites has been examined. The mutation of glutamate to glutamine at position 12 in any of the EF‐hand Ca2+‐binding loops greatly decreases the Ca2+‐binding affinity at that site, yet differs in the overall effects on Ca2+ binding depending on which of the four sites is mutated. When the mutation is in site I, there is only a small decrease in the apparent Ca2+‐binding affinity of site II, and vice versa. Mutation in either site III or IV results in a large decrease in the apparent Ca2+‐binding affinities of the partner C‐terminal site. In both the N‐ and C‐terminal domains, evidence for altered conformational effects in the partners of mutated sites is presented. In the C‐terminus, the conformational consequences of mutating site III or site IV are strikingly different.
Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry
Authors: Papayannopoulos I. A. , Biemann K. 
Published: July 2010

AbstractThe amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.
ββ homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation–renaturation
Authors: Holtzer Marilyn Emerson , Kidd Suzanne Greuel , Crimmins Dan L. , Holtzer Alfred 
Published: July 2010

AbstractNative tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of αα and αβ coiled coils (α/β ≈︁ 3–4/1). In some extant studies, no ββ molecules have been found. In this study, RSTm from several different preparations was disulfide cross‐linked, both preparation and cross‐linking being done under nondenaturing conditions. The cross‐linked product was assayed for the presence of ββ molecules cross‐linked at both C36 and C190 (β=β). In such cross‐linked RSTm, 3–8% β=β is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, C4 reversed‐phase high‐performance liquid chromatography, and a free‐solution capillary electrophoresis experiment. This percentage becomes ∼4–10% ββ when corrected for incomplete double cross‐linking and is independent of protein concentration (0.1–10.0 mg/mL), indicating that the observed ββ species are not artifacts due to intermolecular cross‐linking. Upon denaturation and subsequent renaturation either by heating to 55 °C or by incubating at 45 °C followed by quenching to room temperature, or by guanidine hydrochloride exposure followed by phased renaturation by dialysis, the fraction of ββ increases, indicating that the reassociation favors homodimer formation somewhat over random association. This result differs from the random association observed when the sulfhydryl on one of the chains is carboxyamidomethylated (Holtzer, M.E., Breiner, T., & Holtzer, A., 1984, Biopolymers 23, 1811–1833), and from the overwhelming heterodimer preferences reported for tropomyosins from other organisms (Lehrer, S.S., Qian, Y., & Hvidt, S., 1989, Science 246, 926–928; Lehrer, S.S. & Qian, Y., 1990, J. Biol. Chem. 265, 1134–1138).
Time‐resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: Conformational analysis of intermediates and transition‐state complexes
Authors: Atkins William M. , Villafranca Joseph J. 
Published: July 2010

AbstractSingle tryptophan‐containing mutants of low adenylylation state Escherichia coli glutamine synthetase have been studied by frequency‐domain fluorescence spectroscopy in the presence of various substrates and inhibitors. At pH 6.5, the Mn‐bound wild‐type enzyme (wild type has two tryptophans/subunit) and the mutant enzymes exhibit heterogeneous fluorescence decay kinetics; the individual tryptophans are adequately described by a triple exponential decay scheme. The recovered lifetime values are 5.9 ns, 2.6 ns, and 0.4 ns for Trp‐57 and 5.8 ns, 2.3 ns, and 0.4 ns for Trp‐158. These values are nearly identical to the previously reported results at pH 7.5 (Atkins, W.M., Stayton, P.S., & Villafranca, J.J., 1991, Biochemistry 30, 3406–3416). In addition, Trp‐57 and Trp‐158 both exhibit an ATP‐induced increase in the relative fraction of the long lifetime component, whereas only Trp‐57 is affected by this ligand at pH 7.5. The transition‐state analogue l‐methionine‐(R, S)‐sulfoximine (MSOX) causes a dramatic increase in the fractional intensity of the long lifetime component of Trp‐158. This ligand has no effect on the W158S mutant protein and causes a small increase in the fractional intensity of the long lifetime component of the W158F mutant protein. Addition of glutamate to the ATP complex, which affords the γ‐glutamylphosphate‐ADP complex, results in the presence of new lifetime components at 7, 3.2, and 0.5 ns for Trp‐158, but has no effect on Trp‐57. Similar results were obtained when ATP was added to the MSOX complex; Trp‐57 exhibits heterogeneous fluorescence decay with lifetimes of 7, 3.5, and 0.8 ns. Decay kinetics of Trp‐158 are best fit to a nearly homogeneous decay with a lifetime of 5.5 ns in the MSOX‐ATP inactivated complex. These results provide a model for the sequence of structural and dynamic changes that take place at the Trp‐57 loop and the central loop (Trp‐158) during several intermediate stages of catalysis.
Mast cell tryptases: Examination of unusual characteristics by multiple sequence alignment and molecular modeling
Authors: Johnson David A. , Barton Geoffrey J. 
Published: July 2010

AbstractTryptases are trypsin‐like serine proteinases found in the granules of mast cells. Although they show 40% sequence identity with trypsin and contain only 20 or 21 additional residues, tryptases display several unusual features. Unlike trypsin, the tryptases only make limited cleavages in a few proteins and are not inhibited by natural trypsin inhibitors, they form tetramers, bind heparin, and their activity on synthetic substrates is progressively inhibited as the concentration of salt increases above 0.2 M.Unique sequence features of seven tryptases were identified by comparison to other serine proteinases. The three‐dimensional structures of the tryptases were then predicted by molecular modeling based on the crystal structure of bovine trypsin. The models show two large insertions to lie on either side of the active‐site cleft, suggesting an explanation for the limited activity of tryptases on protein substrates and the lack of inhibition by natural inhibitors. A group of conserved Trp residues and a unique proline‐rich region make two surface hydrophobic patches that may account for the formation of tetramers and/or inhibition with increasing salt. Although they contain no consensus heparin‐binding sequence, the tryptases have 10–13 more His residues than trypsin, and these are positioned on the surface of the model. In addition, clustering of Arg and Lys residues may also contribute to heparin binding. Putative Asn‐linked glycosylation sites are found on the opposite side of the model from the active site. The model provides structural explanations for some to the unusual characteristics of the tryptases and a rational basis for future experiments, such as site‐directed mutagenesis.
Inclusion of solvation free energy with molecular mechanics energy: Alanyl dipeptide as a test case
Authors: Schiffer Celia A. , Caldwell James W. , Stroud Robert M. , Kollman Peter A. 
Published: July 2010

AbstractA combined force field of molecular mechanics and solvation free energy is tested by carrying out energy minimization and molecular dynamics on several conformations of the alanyl dipeptide. Our results are qualitatively consistent with previous experimental and computational studies, in that the addition of solvation energy stabilizes the C5 conformation of the alanyl dipeptide relative to the C7.
An optimization approach to predicting protein structural class from amino acid composition
Authors: Zhang Chun‐Ting , Chou Kuo‐Chen 
Published: July 2010

AbstractProteins are generally classified into four structural classes: all‐α proteins, all‐β proteins, α+β proteins, and α/β proteins. In this article, a protein is expressed as a vector of 20‐dimensional space, in which its 20 components are defined by the composition of its 20 amino acids. Based on this, a new method, the so‐called maximum component coefficient method, is proposed for predicting the structural class of a protein according to its amino acid composition. In comparison with the existing methods, the new method yields a higher general accuracy of prediction. Especially for the all‐α proteins, the rate of correct prediction obtained by the new method is much higher than that by any of the existing methods. For instance, for the 19 all‐α proteins investigated previously by P.Y. Chou, the rate of correct prediction by means of his method was 84.2%, but the correct rate when predicted with the new method would be 100%! Furthermore, the new method is characterized by an explicable physical picture. This is reflected by the process in which the vector representing a protein to be predicted is decomposed into four component vectors, each of which corresponds to one of the norms of the four protein structural classes.
Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry
Authors: Aebersold Ruedi , Bures Edward J. , Goghari Mahesh H. , Namchuk Mark , Shushan Bori , Covey Tom C. 
Published: July 2010

AbstractWe report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion‐evaporation mass spectrometry. We demonstrate that the reagent 3‐[4′(ethylene‐N, N, N‐trimethylamino)phenyl]‐2‐isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion‐evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.
Renaturation of citrate synthase: Influence of denaturant and folding assistants
Authors: Zhi Wang , Landry Samuel J. , Gierasch Lila M. , Srere Paul A. 
Published: July 2010

AbstractCitrate synthase (CS), which has been denatured in either guanidine hydrochloride (GdnHCl) or urea can be assisted in its renaturation in a variety of ways. The addition of each of the assistants – bovine serum albumin (BSA), oxaloacetate (OAA), and glycerol – promotes renaturation. In combination, the effect of these substances is additive with respect to the yield of folded CS. The report of Buchner et al. (Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F.X., & Kiefhaber, T., 1991, Biochemistry 30, 1586–1591) that refolding of CS is facilitated by the GroE system (an Escherichia coli chaperonin [cpn] that is composed of GroEL [cpn60] and GroES [cpn10]) has been confirmed. However, we observed substantially higher yield of reactivated CS, 82%, and almost no reactivation in the absence of GroES, <5%, whereas Buchner et al. reported 28% and 16%, respectively. In addition, we find that GroE‐assisted refolding is more efficient for CS denatured in GdnHCl than for CS denatured in urea. This result is discussed in light of the known difference in the denatured states generated in GdnHCl and urea. Because GroEL inhibits the BSA/glycerol/OAA‐assisted refolding, this system will be useful in future studies on the mechanism of GroE‐facilitated refolding.
Extreme pKa displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes
Authors: Lederer Florence 
Published: July 2010

AbstractFlavocytochrome b2 (or l‐lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related α‐hydroxy acid‐oxidizing FMN‐dependent oxidases. Previous work has shown that, in the active site of the unligated reduced flavocytochrome b2, the group that has captured the substrate α‐proton has a high pKapp, calculated to lie around 15 through the use of Eigen's equation. A detailed inspection of the now known three‐dimensional structure of the enzyme leads to the conclusion that the high pK4 belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction. Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pKa of the reduced FMN N5 position should be lowered by several pH units compared to its pKa of 20 or more when free. The features of the three‐dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion. Finally, it is suggested that similar pK shifts affect the active sites of the α‐hydroxy acid‐oxidizing flavooxidases, which are homologous to flavocytochrome b2. The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.
Characterization of EntF as a serine‐activating enzyme
Authors: Reichert Janice , Sakaitani Masahiro , Walsh Christopher T. 
Published: July 2010

AbstractEntF is the enzyme responsible for serine activation during the biosynthesis of enterobactin (a cyclic trimer of N‐dihydroxybenzoyl serine) in Escherichia coli. EntF has been overexpressed and purified to >90% homogeneity. The enzyme has been shown to complement the entF− MK1 strain in the synthesis of 2,3‐dihydroxybenzoylserine derivatives and exhibits l‐serine‐dependent ATP[32P] pyrophosphate exchange activity with a Km for serine of 260 mM and a turnover number of 760 min−1. Release of PPi during incubation of EntF with serine and ATP was observed, but with a low turnover number of 1.0 min−1. These results suggested the presence of an enzyme‐bound intermediate, which has been shown by gel filtration analysis to be (l‐serine)adenylate.
Convergent evolution of similar enzymatic function on different protein folds: The hexokinase, ribokinase, and galactokinase families of sugar kinases
Authors: Bork Peer , Sander Chris , Valencia Alfonso 
Published: July 2010

AbstractKinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three‐dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three‐dimensional structure, and comprises pro‐ and eukaryotic ribokinases, bacterial fructokinases, the minor 6‐phosphofructokinase 2 from Escherichia coli, 6‐phosphotagatokinase, 1‐phosphofructokinase, and, possibly, inosine‐guanosine kinase. The third family, also of unknown three‐dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases.Each of the three families of sugar kinases appears to have a distinct three‐dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three‐dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.
Disulfide bonding patterns and protein topologies
Authors: Benham Craig J. , Saleet Jafri M. 
Published: July 2010

AbstractThis paper examines the topological properties of protein disulfide bonding patterns. First, a description of these patterns in terms of partially directed graphs is developed. The topologically distinct disulfide bonding patterns available to a polypeptide chain containing n disulfide bonds are enumerated, and their symmetry and reducibility properties are examined. The theoretical probabilities are calculated that a randomly chosen pattern of n bonds will have any combination of symmetry and reducibility properties, given that all patterns have equal probability of being chosen. Next, the National Biomedical Research Foundation protein sequence and Brookhaven National Laboratories protein structure (PDB) databases are examined, and the occurrences of disulfide bonding patterns in them are determined. The frequencies of symmetric and/or reducible patterns are found to exceed theoretical predictions based on equiprobable pattern selection. Kauzmann's model, in which disulfide bonds form during random encounters as the chain assumes random coil conformations, finds that bonds are more likely to form with near neighbor cysteines than with remote cysteines. The observed frequencies of occurrence of disulfide patterns are found here to be virtually uncorrelated with the predictions of this alternative random bonding model. These results strongly suggest that disulfide bond pattern formation is not the result of random factors, but instead is a directed process.Finally, the PDB structure database is examined to determine the extrinsic topologies of polypeptides containing disulfide bonds. A complete survey of all structures in the database found no instances in which two loops formed by disulfide bonds within the same polypeptide chain are topologically linked. Similarly, no instances are found in which two loops present on different polypeptide chains in a structure are catenated. Further, no examples of topologically knotted loops occur. In contrast, pseudolinking has been found to be a relatively frequent event. These results show a complete avoidance of nontrivial topological entanglements that is unlikely to be the result of chance events. A hypothesis is presented to account for some of these observations.