bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cysteinyl peptides labeled by dibromobutanedione in reaction with rabbit muscle pyruvate kinase
Authors: Vollmer Sara H. , Colman Roberta F. 
Published: February 2009

AbstractThe bifunctional reagent 1,4‐dibromobutanedione (DBBD) reacts covalently with pyruvate kinase from rabbit muscle to cause inactivation of the enzyme at a rate that is linearly dependent on the reagent concentration, giving a second order rate constant of 444 min−1 M−1. The individual substrates phosphoenolpyruvate (with KCl), ADP, or ATP in the presence of divalent metal cation provide marked protection against inactivation suggesting that reaction occurs in the region of the active site. The limited incorporation of DBBD into pyruvate kinase was measured by reduction of the carbonyl groups of the enzyme‐bound reagent using [3H]NaBH4. When pyruvate kinase was reacted with 120 μM DBBD at pH 7.0 for 50 min in the absence of protectants, 1.8 mol of tritium/mol of subunit was incorporated, whereas in the presence of phosphoenolpyruvate with KCl, only 1.0 mol of tritium was incorporated per mole of subunit.Modified peptides were isolated from tryptic digests of pyruvate kinase. Reaction of enzyme in the presence of substrate (showing no activity loss) yielded a single peptide, Asn‐Ile‐X1‐Lys, where X1 corresponds to Cys164 of the known amino acid sequence of muscle pyruvate kinase. In the absence of protectants, reaction for 10 min (when the enzyme retained substantial activity) yielded Asn‐Ile‐X1‐Lys as the major labeled peptide, whereas reaction for 50 min (when the enzyme was 88% inactivated) yielded predominantly Asn‐Ile‐X1‐Lys cross‐linked to X2‐Asp‐Glu‐Asn‐Ile‐Leu‐Trp‐Leu‐Asp‐Tyr‐Lys, where X2 corresponds to Cys151. Because activity loss correlates with the appearance of the cross‐linked peptides but not with formation of Asn‐Ile‐X1‐Lys, inactivation is likely caused by the reaction leading to the cross‐link between Cys151 and Cys164. The distance between the α‐carbons of these residues in the crystal structure is 15.5 Å, whereas only 12.0 Å can be spanned by the two side chains linked by a dioxobutyl group, suggesting either that pyruvate kinase undergoes a conformational change in forming the cross‐link or that local rapid fluctuations in structure occur in solution to the extent of 3.5 Å in this region of pyruvate kinase.
A protease‐sensitive site in the proposed Ca2+‐binding region of human serum amyloid P component and other pentraxins
Authors: Kinoshita Carol M. , Gewurz Anita T. , Siegel Joan N. , Ying Shan‐Ching , Gupta Rajesh K. , Huckman Robert , Gewurz Henry , Hugli Tony E. , Coe John E. 
Published: February 2009

AbstractSerum amyloid P component (SAP) is a decamer of 10 identical 25.5‐kDa subunits. Limited proteolysis of SAP with α‐chymotrypsin cleaves the subunit into two fragments of 18 and 7.5 kDa, although the fragments stay together in the decamer under nondenaturing conditions. Proteolysis does not occur in the presence of Ca2+ (10 mM). Cleavage with α‐chymotrypsin prevents the Ca2+‐dependent binding of SAP to zymosan extract, nucleosomes, and DNA. The α‐chymotrypsin cleavage site identified is in a region of SAP that is highly conserved in members of the human C‐reactive protein (CRP) family of proteins (pentraxins) to which SAP belongs and is similar to the Ca2+‐binding site in calmodulin and related Ca2+‐binding proteins (Nguyen, N.Y., Suzuki, A., Boykins, R.A., & Liu, T.‐Y., 1986, J. Biol. Chem. 261, 10456–10465). Treatment of SAP with other proteases (trypsin, Pronase, and Nagarse protease) yields fragmentation patterns upon sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) that are similar to those obtained with α‐chymotrypsin. Two other members of the pentraxin family of proteins, hamster female protein and rabbit CRP, also exhibit similar fragmentation patterns on SDS‐PAGE when treated with the various proteases. Recently, it has been shown that the homologous protein, human CRP, is cleaved in the same homologous position as cleavage of SAP by α‐chymotrypsin, resulting in the loss of Ca2+ binding (as shown by equilibrium dialysis) and Ca2+‐dependent binding reactivities (Kinoshita, C.M., Ying, S.‐C., Hugli, T.E., Siegel, J.N., Potempa, L.A., Jiang, H.J., Houghten, R.A., & Gewurz, H., 1989, Biochemistry 28, 9840–9848). These results indicate that the protease sensitivity of this proposed Ca2+‐binding region has been conserved and may play an important regulatory role, perhaps via the control of Ca2+‐dependent properties of these proteins.
Structural and functional analyses of the repressor, RbsR, of the ribose operon of Escherichia coli
Authors: Mauzy C.A. , Hermodson M.A. 
Published: February 2009

AbstractThe DNA sequence encoding the rbs repressor protein, RbsR, has been determined. Amino acid sequence analyses of the product of an rbsR‐lacZ fusion and of affinity‐purified RbsR demonstrate that translation begins at an unusual codon, TTG, and that the initial amino acid is removed during maturation of the protein. DNA‐binding assays indicate that RbsR binds to a region of perfect dyad symmetry spanning the rbs operon transcriptional start site and that the affinity for the rbs operator is reduced by addition of ribose, consistent with ribose being the inducer of the operon. RbsR is a member of a family of homologous repressor proteins having very similar DNA‐binding sites and binding to similar operator sequences. [RbsR PIR accession number A41828.]
Cis proline mutants of ribonuclease A. II. Elimination of the slow‐folding forms by mutation
Authors: Schultz David A. , Baldwin Robert L. , Schmid Franz X. 
Published: February 2009

AbstractRibonuclease A is known to form an equilibrium mixture of fast‐folding (UF) and slow‐folding (US) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, USII, USI, and possibly also minor populations of other US species. The two cis proline residues, P93 and P114, are logical candidates for producing the major US species after unfolding, by slow cis → trans isomerization. Much work has been done in the past on testing this proposal, but the results have been controversial. Site‐directed mutagenesis is used here. Four single mutants, P93A, P93S, P114A, and P114G, and also the double mutant P93A, P114G have been made and tested for the formation of US species after unfolding. The single mutants P114G and P114A still show slow isomerization reactions after unfolding that produce US species; thus, Pro 114 is not required for the formation of at least one of the major US species of ribonuclease A. Both the refolding kinetics and the isomerization kinetics after unfolding of the Pro 93 single mutants are unexpectedly complex, possibly because the substituted amino acid forms a cis peptide bond, which should undergo cis → trans isomerization after unfolding. The kinetics of peptide bond isomerization are not understood at present and the Pro 93 single mutants cannot be used yet to investigate the role of Pro 93 in forming the US species of ribonuclease A.The double mutant P93A, P114G shows single exponential kinetics measured by CD, and it shows no evidence of isomerization after unfolding. Thus, the unfolding and refolding kinetics of the double mutant indicate that replacement of Pro 93 and Pro 114 has removed the residues responsible for forming the major US species. This result, taken together with data for the Pro 114 single mutants, indicates that Pro 93 and Pro 114 are collectively responsible for forming the major US species.
Inhibitory effects of HSP70 chaperones on nascent polypeptides
Authors: Ryan Christine , Schlesinger Milton J. , Stevens Tom H. 
Published: February 2009

AbstractSeveral of the major heat shock proteins (HSPs) function normally as molecular chaperones to prevent aggregation of immature polypeptides and thereby facilitate folding and oligomerization. To determine their effect on nascent polypeptides, we added purified preparations of different isoforms of HSP70 to in vitro translation reactions primed by the 26S mRNA of Sindbis virus, which encodes an autoprotease that functions cotranslationally, or by the mRNA encoding the yeast vacuolar H+ATPase, which is formed by a novel transpeptidase activity that removes the central region of the initial polypeptide. In the presence of HSP70s both the autoprotease and transpeptidase activities were inhibited, indicating that these chaperones can interact with nascent polypeptides and, in the cases studied here, perturb their normal structures.
Multiple phosphate positions in the catalytic site of glycogen phosphorylase: Structure of the pyridoxal‐5′‐pyrophosphate coenzyme‐substrate analog
Authors: Sprang Stephen R. , Madsen Neil B. , Withers Stephen G. 
Published: February 2009

AbstractThe three‐dimensional structure of an R‐state conformer of glycogen phosphorylase containing the coenzyme‐substrate analog pyridoxal‐5′‐diphosphate at the catalytic site (PLPP‐GPb) has been refined by X‐ray crystallography to a resolution of 2.87 Å. The molecule comprises four subunits of phosphorylase related by approximate 222 symmetry. Whereas the quaternary structure of R‐state PLPP‐GPb is similar to that of phosphorylase crystallized in the presence of ammonium sulfate (Barford, D. & Johnson, L.N., 1989, Nature 340, 609–616), the tertiary structures differ in that the two domains of the PLPP‐GPb subunits are rotated apart by 5° relative to the T‐state conformation. Global differences among the four subunits suggest that the major domains of the phosphorylase subunit are connected by a flexible hinge. The two different positions observed for the terminal phosphate of the PLPP are interpreted as distinct phosphate subsites that may be occupied at different points along the reaction pathway. The structural basis for the unique ability of R‐state dimers to form tetramers results from the orientation of subunits with respect to the dyad axis of the dimer. Residues in opposing dimers are in proper registration to form tetramers only in the R‐state.
Avian 3‐hydroxy‐3‐methylglutaryl‐CoA lyase: Sensitivity of enzyme activity to thiol/disulfide exchange and identification of proximal reactive cysteines
Authors: Hruz Paul W. , Miziorko Henry M. 
Published: February 2009

AbstractCatalysis by purified avian 3‐hydroxy‐3‐methylglutaryl‐CoA lyase is critically dependent on the reduction state of the enzyme, with less than 1% of optimal activity being observed with the air‐oxidized enzyme. The enzyme is irreversibly inactivated by sulfhydryl‐directed reagents with the rate of this inactivation being highly dependent upon the redox state of a critical cysteine. Methylation of reduced avian lyase with 1 mM 4‐methylnitrobenzene sulfonate results in rapid inactivation of the enzyme with a kinact of 0.178 min−1. The oxidized enzyme is inactivated at a sixfold slower rate (kinact = 0.028 min−1). Inactivation of the enzyme with the reactive substrate analog 2‐butynoyl‐CoA shows a similar dependence upon the enzyme's redox state, with a sevenfold difference in kinact observed with oxidized vs. reduced forms of the enzyme. Chemical cross‐linking of the reduced enzyme with stoichiometric amounts of the bifunctional reagents 1,3‐dibromo‐2‐propanone (DBP) or N, N′‐ortho‐phenylenedimaleimide (PDM) coincides with rapid inactivation. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of enzyme treated with bifunctional reagent reveals a band of twice the molecular weight of the lyase monomer, indicating that an intersubunit cross‐link has been formed. Differential labeling of native and cross‐linked protein with [1‐14C]iodoacetate has identified as the primary cross‐linking target a cysteine within the sequence VSQAACR, which maps at the carboxy‐terminus of the cDNA‐deduced sequence of the avian enzyme (Mitchell, G.A., et al., 1991, Am. J. Hum. Genet. 49, 101). In contrast, bacterial HMG‐CoA lyase, which contains no corresponding cysteine, is not cross‐linked by comparable treatment with bifunctional reagent. These results provide evidence for a potential regulatory mechanism for the eukaryotic enzyme via thiol/disulfide exchange and identify a cysteinyl residue with the reactivity and juxtaposition required for participation in disulfide formation.
Three‐dimensional structure of the Fab fragment of a neutralizing antibody to human rhinovirus serotype 2
Authors: Tormo José , Fita Ignasi , Stadler Elisabeth , Skern Tim , Auer Herbert , Blaas Dieter , Kanzler Otto , Betzel Christian 
Published: February 2009

AbstractThe crystal structure of the antigen‐binding fragment of a monoclonal antibody (8F5) that neutralizes human rhinovirus serotype 2 has been determined by X‐ray diffraction studies. Antibody 8F5, obtained by immunization with native HRV2 virions, cross‐reacts with peptides of the viral capsid protein VP2, which contribute to the neutralizing immunogenic site B in this serotype. The structure was solved by the molecular replacement method and has been refined to an R‐factor of 18.9% at 2.8 Å resolution. The elbow angle, relating the variable and constant modules of the molecule is 127°, representing the smallest elbow angle observed so far in an Fab fragment. Furthermore, the charged residues of the epitope can be well accommodated in the antigen‐binding site. This is the first crystal structure reported for an antibody directed against an icosahedral virus.
Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli
Authors: Wang Cheng C. , Moriyama Ryuichi , Low Philip S. , Badylak Janine A. , Dixon Jack E. , Lux Samuel E. 
Published: February 2009

AbstractThe cytoplasmic domain of the human erythrocyte membrane protein, band 3 (cdb3), contains binding sites for hemoglobin, several glycolytic enzymes, band 4.1, band 4.2, and ankyrin, and constitutes the major linkage between the membrane skeleton and the membrane. Although erythrocyte cdb3 has been partially purified from proteolyzed red blood cells, further separation of the water‐soluble 43‐kDa and 41‐kDa proteolytic fragments has never been achieved. In order to obtain pure cdb3 for crystallization and site‐directed mutagenesis studies, we constructed an expression plasmid that has a tandemly linked T7 promoter placed upstream of the N‐terminal 379 amino acids of the erythrocyte band 3 gene. Comparison of several Escherichia coli strains led to the selection of the BL21 (DE3) strain containing the pLysS plasmid as the best host for efficient production of cdb3. About 10 mg of recombinant cdb3 can be easily purified from 4 L of E. coli culture in two simple steps. Comparison of cdb3 released from the red blood cell by proteolysis with recombinant cdb3 reveals that both have the same N‐terminal sequence, secondary structure, and pH‐dependent conformational change. The purified recombinant cdb3 is also a soluble stable dimer with the same Stokes radius as erythrocyte cdb3. The affinities of the two forms of cdb3 for ankyrin are essentially identical; however, recombinant cdb3 with its unblocked N‐terminus exhibits a slightly lower affinity for aldolase.
Synthetic “interface” peptides alter dimeric assembly of the HIV 1 and 2 proteases
Authors: Babé Lilia M. , Rosé Jason , Craik Charles S. 
Published: February 2009

AbstractRetroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N‐ and C‐terminal residues of both monomers, which form a four‐stranded β‐sheet. Peptides mimicking one β‐strand (residues 95–99), or two β‐strands (residues 1–5 plus 95–99 or 95–99 plus 95–99) from the human immunodeficiency virus 1 (HIV1) interface were shown to inhibit the HIV1 and 2 proteases (PRs) with IC50's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIV1 PR dimer was not inhibited to the same extent as the wild‐type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two‐subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one‐strand peptide causes a shift in the observed Kd for the HIV1 PR. Both one‐ and two‐strand peptides alter the monomer/dimer equilibrium of both HIV1 and HIV2 PRs. This was shown by the reduced cross‐linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIV1 and HIV2 PRs with the interface peptides shows that only the two‐strand peptides prevent the assembly of active PR dimers. Although both one‐ and two‐strand peptides seem to affect dimer dissociation, only the two‐strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.
On the multiple simultaneous superposition of molecular structures by rigid body transformations
Authors: Diamond R. 
Published: February 2009

AbstractA method of optimally superimposing n coordinate sets on each other by rigid body transformations, which minimizes the sum of all n(n ‐ 1)/2 pairwise residuals, is presented. In the solution phase the work load is approximately linear on n, is independent of the size of the structures, is independent of their initial orientations, and terminates in one cycle if n = 2 or if the coordinate sets are exactly superposable, and otherwise takes a number of cycles dependent only on genuine shape differences. Enantiomorphism, if present, is detected, in which case the option exists to reverse or not to reverse the chirality of relevant coordinate sets. The method also offers a rational approach to the problem of multiple minima and has successfully identified four distinct minima in such a case. Source code, which is arranged to enable the study of the disposition of domains in multidomain structures, is available from the author.
Specificity mapping of cellulolytic enzymes: Classification into families of structurally related proteins confirmed by biochemical analysis
Authors: Claeyssens Marc , Henrissat Bernard 
Published: February 2009

AbstractThe specificities of 15 cellulolytic enzymes have been examined using chromophoric glycosides derived from d‐glucose, cellobiose, higher cellooligosaccharides, lactose, d‐xylose, and β‐(1,4)‐xylobiose. Coinciding with a classification based on hydrophobic cluster analysis of amino acid sequences, six families each showing a characteristic specificity pattern were observed. Furthermore, in these cases where the anomeric forms of reaction products were determined, results seem to indicate conservation of intrinsic reaction mechanism (single or double displacement) within each family. On the other hand, the low molecular weight substrates do not discriminate exo‐ from endocellulases. This functional differentiation is speculated to originate from the presence, in exoenzymes, of a tunnel‐shaped active site formed by extra loops in their structure.
The calmodulin‐binding site of the plasma membrane Ca2+ pump interacts with the transduction domain of the enzyme
Authors: Falchetto Rocco , Vorherr Thomas , Carafoli Ernesto 
Published: February 2009

AbstractCalpain proteolysis of the plasma membrane Ca2+ pump removes a C‐terminal 14‐kDa portion which includes the calmodulin‐binding domain. This produces a fully activated 124‐kDa fragment, which can be inhibited by synthetic versions of the calmodulin‐binding domain. The inhibition is strongest when Trp‐8 in the latter domain is replaced by a Tyr residue (Falchetto, R., Vorherr, T., Brunner, J., & Carafoli, E., 1991, J. Biol. Chem. 266, 2930–2936). In the present study, the N‐terminus of the 28‐residue synthetic calmodulin‐binding domain was acetylated with 3H‐acetic anhydride, and Phe in position 25 was replaced by a phenylalanine derivatized with a diazirine‐based, photoactivatable carbene precursor. This peptide (C28WC*) inhibited the fully active 124‐kDa fragment of the pump and became cross‐linked to it upon photolysis. After proteolysis of the fragment with Asp‐N or Staphylococcus aureus V8 (Glu‐C) protease, labeled peptides were isolated by reversed‐phase high‐performance liquid chromatography and subjected to Edman sequence analysis. The peptides originated from a region of the pump located within the unit protruding into the cytoplasm between transmembrane domain two and three. This unit has been proposed to be the site of the energy transduction domain, which would couple the ATP hydrolysis to Ca2+ translocation.
Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129
Authors: Violand B.N. , Schlittler M.R. , Kolodziej E.W. , Toren P.C. , Cabonce M.A. , Siegel N.R. , Duffin K.L. , Zobel J.F. , Smith C.E. , Tou J.S. 
Published: February 2009

AbstractAspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed‐phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126–133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.
Constraints imposed by protease accessibility on the trans‐membrane and surface topography of the colicin E1 ion channel
Authors: Zhang Yan‐Liang , Cramer William A. 
Published: February 2009

AbstractThe surface topography of a 190‐residue COOH‐terminal colicin E1 channel peptide (NH2‐Met 333‐Ile 522‐COOH) bound to uniformly sized 0.2‐μm liposomes was probed by accessibility of the peptide to proteases in order (1) to determine whether the channel structure contains trans‐membrane segments in addition to the four α‐helices previously identified and (2) to discriminate between different topographical possibilities for the surface‐bound state. An unfolded surface‐bound state is indicated by increased trypsin susceptibility of the bound peptide relative to that of the peptide in aqueous solution. The peptide is bound tightly to the membrane surface with Kd < 10−7 M. The NH2‐terminal 50 residues of the membrane‐bound peptide are unbound or loosely bound as indicated by their accessibility to proteases, in contrast with the COOH‐terminal 140 residues, which are almost protease inaccessible. The general protease accessibility of the NH2‐terminal segment Ala 336‐Lys 382 excludes any model for the closed channel state that would include trans‐membrane helices on the NH2‐terminal side of Lys 382. Lys 381‐Lys 382 is a major site for protease cleavage of the surface‐bound channel peptide. A site for proteinase K cleavage just upstream of the amphiphilic gating hairpin (K420‐K461) implies the presence of a surface‐exposed segment in this region. These protease accessibility data indicate that it is unlikely that there are any α‐helices on the NH2‐terminal side of the gating hairpin K420‐K461 that are inserted into the membrane in the absence of a membrane potential. A model for the topography of an unfolded monomeric surface‐bound intermediate of the colicin channel domain, including a trans‐membrane hydrophobic helical hairpin and two or three long surface‐bound helices, is proposed.