bluuebunny/crossref_metadata_2025_split huggingface dataset:
Control of nucleation in the crystallization of lysozyme
Authors: Chayen Naomi E. , Radcliffe Jonathan W. , Blow David M. 
Published: July 2010

AbstractThis work investigates the influence of storage of lysozyme in solution on its crystallization. The crystallization of hen egg‐white lysozyme exhibits a storage effect (aging) that depends on the length of time the lysozyme solution is stored, after dissolving from freeze‐dried powder, before being brought to crystallization conditions. The number of crystals obtained increases, while their size decreases, as the solution ages. Observations suggest that this effect is due to the presence of fungi that multiply in the stored protein solution. This aging effect was used to control nucleation and determine the number and size of lysozyme crystals to be formed in a given sample.
Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c‐type cytochrome
Authors: Chen Longyin , Mathews F. Scott , Davidson Victor L. , Tegoni Mariella , Rivetti Claudio , Rossi Gian Luigi 
Published: July 2010

AbstractA ternary electron transfer protein complex has been crystallized and a preliminary structure investigation has been carried out. The complex is composed of a quinoprotein, methylamine dehydrogenase (MADH), a blue copper protein, amicyanin, and a c‐type cytochrome (c551i). All three proteins were isolated from Paracoccus denitrificans. The crystals of the complex are orthorhombic, space group C2221 with cell dimensions a = 148.81 Å, b = 68.85 Å, and c = 187.18 Å. Two types of isomorphous crystals were prepared: one using native amicyanin and the other copper‐free apo‐amicyanin. The diffraction data were collected at 2.75 Å resolution from the former and at 2.4 Å resolution from the latter. The location of the MADH portion was determined by molecular replacement. The copper site of the amicyanin molecule was located in an isomorphous difference Fourier while the iron site of the cytochrome was found in an anomalous difference Fourier. The MADH from P. denitrificans (PD‐MADH) is an H2L2 hetero‐tetramer with the H subunit containing 373 residues and the L subunit 131 residues, the latter containing a novel redox cofactor, tryptophan tryptophylquinone (TTQ). The amicyanin of P. denitrificans contains 105 residues and the cytochrome c551i contains 155 residues. The ternary complex consists of one MADH tetramer with two molecules of amicyanin and two of c551i, forming a hetero‐octamer; the octamer is located on a crystallographic diad. The relative positions of the three redox centers — i.e., the TTQ of MADH, the copper of amicyanin, and the heme group of c551i — are presented.
Mapping antibody binding sites on cytochrome c with synthetic peptides: Are results representative of the antigenic structure of proteins?
Authors: Schwab Christian , Twardek Andrea , Bosshard Hans Rudolf , Lo Terence P. , Brayer Gary D. 
Published: July 2010

AbstractCrystallographic work on antigen‐antibody complexes has revealed that extensive surface areas of proteins may interact with antibodies. On the other hand, most experimental approaches to locate and define antigenic determinants of protein antigens rely on the linear sequence of the polypeptide chain. Hence the question arises whether mapping of antibody binding sites by analysis of the reactivity of anti‐protein antibodies with synthetic peptides can provide a representative picture of the antigenic structure of a protein antigen. We have addressed this question using yeast iso‐1 cytochrome c as a protein antigen against which antisera were raised in rabbits. The reaction of the antisera with 103 synthetic hexapeptides covering the entire sequence of cytochrome c was tested by the pepscan procedure in which peptides are coupled to polyethylene rods and tested by ELISA. For the assay, anti‐cytochrome c antibodies were fractionated by affinity chromatography on native yeast iso‐1 cytochrome c and on apo‐cytochrome c; the latter is a random coil. It was found that only antibodies retained by the apocytochrome c affinity column react with synthetic peptides. These antibodies comprise a small fraction, probably less than 2%, of all cytochrome c‐specific antibodies. The majority of antigenic determinants, which seem to consist of strongly conformation‐dependent topographic epitopes, could not be uncovered by the peptide approach. Epitope mapping with short peptides seems of limited usefulness in the case of small, globular, and conformationally stable proteins like cytochrome c.
Modulation of antibody affinity by a non‐contact residue
Authors: Schildbach Joel F. , Near Richard I. , Bruccoleri Robert E. , Haber Edgar , Jeffrey Philip D. , Novotny Jiri , Sheriff Steven , Margolies Michael N. 
Published: July 2010

AbstractAntibody LB4, produced by a spontaneous variant of the murine anti‐digoxin monoclonal antibody 26–10, has an affinity for digoxin two orders of magnitude lower than that of the parent antibody due to replacement of serine with phenylalanine at position 52 of the heavy chain variable region (Schildbach, J.F., Panka, D.J., Parks, D.R., et al., 1991, J. Biol. Chem. 266, 4640–4647). To examine the basis for the decreased affinity, a panel of engineered antibodies with substitutions at position 52 was created, and their affinities for digoxin were measured. The antibody affinities decreased concomitantly with increasing size of the substituted side chains, although the shape of the side chains also influenced affinity. The crystal structure of the 26–10 Fab complexed with digoxin (P.D.J., R.K. Strong, L.C. Sieker, C. Chang, R.L. Campbell, G.A. Petsko, E.H., M.N.M., & S.S., submitted for publication) shows that the serine at heavy chain position 52 is not in contact with hapten, but is adjacent to a tyrosine at heavy chain position 33 that is a contact residue. The mutant antibodies were modeled by applying a conformational search procedure to position side chains, using the 26–10 Fab crystal structure as a starting point. The results suggest that each of the substituted side chains may be accommodated within the antibody without substantial structural rearrangement, and that none of these substituted side chains are able to contact hapten. These modeling results are consistent with the substituents at position 52 having only an indirect influence upon antibody affinity. The mutagenesis and modeling results suggest that even conservative replacements of non‐contact residues can alter affinity indirectly through their impact on contact residue placement.
A study of intermediates involved in the folding pathway for recombinant human macrophage colony‐stimulating factor (M‐CSF): Evidence for two distinct folding pathways
Authors: Wilkins James A. , Cone James , Randhawa Zafar I. , Wood David , Warren M. Kim , Witkowska H. Ewa 
Published: July 2010

AbstractThe folding pathway for a 150‐amino acid recombinant form of the dimeric cytokine human macrophage colony‐stimulating factor (M‐CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non‐native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M‐CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell‐based assay. The results presented here strongly suggest that M‐CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.
Mannose‐specific lectins bind α‐2‐macroglobulin and an unknown protein from human plasma
Authors: Van Leuven F. , Torrekens S. , Van Berghe H. Den , Van Damme E. , Peumans W. 
Published: July 2010

AbstractGNA, the mannose‐specific lectin from Galanthus nivalis was confirmed to bind α‐2‐macroglobulin (A2M) but another protein was copurified with A2M from total human plasma. A total of 23 other lectins with diverse specificities were tested for reaction with human A2M and with three other members of the A2M family. NPA, a mannose‐specific lectin isolated from Narcissus pseudonarcissus bulbs, and RSA, the Rhizoctonia solani agglutinin, were selected for further testing. For isolation of A2M, immobilized NPA was superior to GNA because its binding capacity was an order of magnitude higher. The specificity of these lectins must be very similar however, because the same unknown plasma protein was also bound by NPA. A2M and the unknown protein must share a unique mannose carbohydrate structure not present in any other human plasma protein. The copurified protein subunit size of 185 kDa is very similar to that of A2M, but the native molecular mass of 350 kDa indicated a noncovalent homodimer structure. Together with the acid isoelectric point this is not typical for any known plasma protein nor for any unidentified spot on the two‐dimensional map of human plasma proteins. No immunological reaction with available antisera was evident. A specific antiserum raised to the unknown protein demonstrated its presence in all human plasma samples examined. The N‐terminal residue was blocked, whereas internal protein sequences obtained after CNBr fragmentation and proteolysis were not homologous to any known protein sequence. These data demonstrate that this protein is unknown and not a proteinase inhibitor of the A2M family.
Relationship between sequence conservation and three‐dimensional structure in a large family of esterases, lipases, and related proteins
Authors: Cygler Miroslaw , Schrag Joseph D. , Sussman Joel L. , Harel Michal , Silman Israel , Gentry Mary K. , Doctor Bhupendra P. 
Published: July 2010

AbstractBased on the recently determined X‐ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three‐dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X‐ray structures.
Packing and hydrophobicity effects on protein folding and stability: Effects of β‐branched amino acids, valine and isoleucine, on the formation and stability of two‐stranded α‐helical coiled coils/leucine zippers
Authors: Zhu Bing‐Yan , Zhou Man E. , Kay Cyril M. , Hodges Robert S. 
Published: July 2010

AbstractThe aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three‐dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA‐binding proteins (leucine zippers), Leu residues are conserved at positions “d,” and β‐branched amino acids, Ile and Val, often occur at positions “a” in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two‐stranded α‐helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions “a” (9, 16, and 23) or “d” (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn·l. The results of the mutations of Ile to Val at either positions “a” or “d” in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (δδG values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions “a” in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (δδG values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic α‐helices. These structural findings correlate with the observation that in DNA‐binding proteins, the conserved Leu residues at positions “d” are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions “a.”
Structure of recombinant ricin A chain at 2.3 Å
Authors: Mlsna Debra , Monzingo Arthur F. , Katzin Betsy J. , Ernst Stephen , Robertus Jon D. 
Published: July 2010

AbstractThe plant cytotoxin ricin is a heterodimer with a cell surface binding (B) chain and an enzymatically active A chain (RTA) known to act as a specific N‐glycosidase. RTA must be separated from B chain to attack rRNA. The X‐ray structure of ricin has been solved recently; here we report the structure of the isolated A chain expressed from a clone in Escherichia coli. This structure of wild‐type rRTA has and will continue to serve as the parent compound for difference Fouriers used to assess the structure of site‐directed mutants designed to analyze the mechanism of this medically and commercially important toxin. The structure of the recombinant protein, rRTA, is virtually identical to that seen previously for A chain in the heterodimeric toxin. Some minor conformational changes due to interactions with B chain and to crystal packing differences are described. Perhaps the most significant difference is the presence in rRTA of an additional active site water. This molecule is positioned to act as the ultimate nucleophile in the depurination reaction mechanism proposed by Monzingo and Robertus (1992, J. Mol. Biol. 227, 1136–1145).
Peptidylglycine α‐amidating monooxygenase: A multifunctional protein with catalytic, processing, and routing domains
Authors: Eipper Betty A. , Milgram Sharon L. , Jean Husten E. , Yun Hye‐Young , Mains Richard E. 
Published: July 2010

AbstractPeptide α‐amidation is a widespread, often essential posttranslational modification shared by many bioactive peptides and accomplished by the products of a single gene encoding a multifunctional protein, peptidylglycine α‐amidating monooxygenase (PAM). PAM has two catalytic domains that work sequentially to produce the final α‐amidated product peptide. Tissue‐specific alternative splicing can generate forms of PAM retaining or lacking a domain required for the posttranslational separation of the two catalytic activities by endoproteases found in neuroendocrine tissue. Tissue‐specific alternative splicing also governs the presence of a transmembrane domain and generation of integral membrane or soluble forms of PAM. The COOH‐terminal domain of the integral membrane PAM proteins contains routing information essential for the retrieval of PAM from the surface of endocrine and nonendocrine cells. Tissue‐specific endoproteolytic processing can generate soluble PAM proteins from integral membrane precursors. Soluble PAM proteins are rapidly secreted from stably transfected nonneuroendo‐crine cells but are stored in the regulated secretory granules characteristic of neurons and endocrine cells.
Sequence analyses and evolutionary relationships among the energy‐coupling proteins enzyme I and HPr of the bacterial phosphoenolpyruvate: Sugar phosphotransferase system
Authors: Reizer Jonathan , Hoischen Christian , Reizer Aiala , Pham Tam N. , Saier Milton H. 
Published: July 2010

AbstractWe have previously reported the overexpression, purification, and biochemical properties of the Bacillus subtilis Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (Reizer, J., et al., 1992, J. Biol. Chem. 267, 9158–9169). We now report the sequencing of the ptsl gene of B. subtilis encoding Enzyme I (570 amino acids and 63, 076 Da). Putative transcriptional regulatory signals are identified, and the pts operon is shown to be subject to carbon source‐dependent regulation. Multiple alignments of the B. subtilis Enzyme I with (1) six other sequenced Enzymes I of the PTS from various bacterial species, (2) phosphoenolpyruvate synthase of Escherichia coli, and (3) bacterial and plant pyruvate:phosphate dikinases (PPDKs) revealed regions of sequence similarity as well as divergence. Statistical analyses revealed that these three types of proteins comprise a homologous family, and the phylogenetic tree of the 11 sequenced protein members of this family was constructed. This tree was compared with that of the 12 sequenced HPr proteins or protein domains. Antibodies raised against the B. subtilis and E. coli Enzymes I exhibited immunological cross‐reactivity with each other as well as with PPDK of Bacteroides symbiosus, providing support for the evolutionary relationships of these proteins suggested from the sequence comparisons. Putative flexible linkers tethering the N‐terminal and the C‐terminal domains of protein members of the Enzyme I family were identified, and their potential significance with regard to Enzyme I function is discussed. The codon choice pattern of the B. subtilis and E. coli ptsI and ptsH genes was found to exhibit a bias toward optimal codons in these organisms. Quantitative analysis of this bias indicated that the ptsH genes have higher bias toward codons favored in highly expressed genes than the ptsI genes, in agreement with their relative levels of expression.
Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated IIIGlc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques
Authors: Pelton J.G. , Torchia D.A. , Meadow N.D. , Roseman S. 
Published: July 2010

AbstractIIIGlc is an 18.1‐kDa signal‐transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two‐dimensional 1H‐15N and 1H‐13C heteronuclear multiple‐quantum coherence (HMQC) experiments and a two‐dimensional 13C‐13C‐1H correlation spectroscopy via JCC coupling experiment. The data were acquired on uniformly 15N‐labeled and uniformly 15N/13C‐labeled protein samples. The experiments rely on one‐bond and two‐bond J couplings that allowed for assignment of the signals without the need for the analysis of through‐space (nuclear Overhauser effect spectroscopy) correlations. The 15N and 13C chemical shifts were used to determine that His‐75 exists predominantly in the Nε2‐H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His‐90 exists primarily in the Nδ1‐H state in the unphosphorylated protein. Upon phosphorylation of the Nε2 nitrogen of His‐90, the Nδ1 nitrogen remains protonated, resulting in the formation of a charged phospho‐His‐90 moiety. The 1H, 15N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pKa values for both His‐75 and His‐90 in IIIGlc and His‐75 in phospho‐IIIGlc are less than 5.0, and that the pKa value for phospho‐His‐90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed.
Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry
Authors: Tanaka Akiyoshi , Sturtevant Julian M. , Flanagan John 
Published: July 2010

AbstractThe effects of eight mutations on the thermodynamics of the reversible thermal unfolding of staphylococcal nuclease have been determined over a range of pH and protein concentration by means of differential scanning calorimetry. Variation of the protein concentration was included in our study because we found a significant dependence of the thermodynamics of protein unfolding on concentration. Values for the change in the standard free energy of unfolding, ΔΔGd0, produced by the mutations in the pH range 5.0–7.0 varied from 1.9 kcal mol−1 (apparent stabilization) for H124L to ‐2.8 kcal mol−1 (apparent destabilization) for L25A. As has been observed in numerous other cases, there is no correlation in magnitude or sign between ΔΔGd0 and the corresponding values for ΔΔHd and TΔΔSd0, the latter quantities being in most cases much larger in magnitude than ΔΔGd0. This fact emphasizes the difficulty in attempting to correlate the thermodynamic changes with structural changes observed by X‐ray crystallography.
Crevice‐forming mutants in the rigid core of bovine pancreatic trypsin inhibitor: Crystal structures of F22A, Y23A, N43G, and F45A
Authors: Danishefsky Avis T. , Housset Dominique , Wlodawer Alexander , Kim Key‐Sun , Tao Feng , Fuchs James , Woodward Clare 
Published: July 2010

AbstractCrystal structures of four mutants of bovine pancreatic trypsin inhibitor (F22A, Y23A, N43G, and F45A), engineered to alter their stability properties, have been determined. The mutated residues, which are highly conserved among Kunitz‐type inhibitors, are located in the rigid core of the molecule. Replacement of the partially buried bulky residues of the wild‐type protein with smaller residues resulted in crevices open to the exterior of the molecule. The overall three‐dimensional structure of these mutants is very similar to that of the wild‐type protein and only small rearrangements are observed among the atoms lining the crevices.