bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rational design of a three‐heptad coiled‐coil protein and comparison by molecular dynamics simulation with the GCN4 coiled coil: Presence of interior three‐center hydrogen bonds
Authors: Rozzelle James E. , Tropsha Alexander , Erickson Bruce W. 
Published: July 2010

Abstractα‐Helical coiled coils have a 7‐residue repeating pattern (abcdefg) where a and d are usually hydrophobic. We have designed a 2‐stranded 44‐residue coiled‐coil protein (P44) consisting of 222‐residue α‐helices linked by 2 terminal disulfide groups to test whether the disulfide bridges could stabilize a 3‐heptad coiled coil. P44 should be stabilized by intrahelical hydrogen bonds, interhelical disulfide and salt bridges, and interior hydrophobic interactions. A computer model of P44 was built and its stability was studied by molecular dynamics simulation with explicit water. This doubly crosslinked 3‐heptad coiled coil did not unfold during a 300‐ps simulation. But reduced P44 with 4 thiol groups did unfold. For comparison, the 62‐residue crystal structure of the 4‐heptad coiled coil of transcription activator GCN4 did not unfold during a 300‐ps simulation. Thus P44 may be a stable folded protein in aqueous solution. These simulations revealed the presence of 2 local hydrogen bond networks involving intrahelical 3‐center hydrogen bonds in the hydrophobic interior of the coiled coils of GCN4 and P44. The NH hydrogen at d makes a 3‐center hydrogen bond whose major component is to the i — 4 C=O oxygen at g and minor component is to the solvent‐inaccessible i — 3 C=O oxygen at a. Likewise, the NH hydrogen at g makes a 3‐center hydrogen bond with the i — 4 C=O oxygen at c and the buried i — 3 C=O oxygen at d.
Conserved sequence pattern in a wide variety of phosphoesterases
Authors: Koonin Eugene V. 
Published: July 2010

AbstractA unique sequence pattern, designated the GD/GNH signature, was shown to be conserved in a wide variety of phosphoesterases. The enzymes containing this signature cleave phosphoester bonds in such different substrates as (1) phosphoserine and phosphothreonine in polypeptides; (2) bis(5′‐nucleosidyl)‐tetraphosphates; (3) nucleoside 5′ phosphates; (4) 2′,3′‐cyclic nucleotide phosphates; (5) polynucleotides; (6) 2′‐5′ phospho‐diesters in RNA (intron) lariats; (7) sphingomyelin; and (7) various phosphomonoesters. Two conserved acidic amino acid residues and a conserved histidine residue may be directly involved in phosphoester bond cleavage.
The effect of N‐terminal acetylation on the structure of an N‐terminal tropomyosin peptide and αα‐tropomyosin
Authors: Greenfield Norma J. , Stafford Walter E , Hitchcock‐Degregori Sarah E. 
Published: July 2010

AbstractWe have used a synthetic peptide consisting of the first 30 residues of striated muscle α‐tropomyosin, with GlyCys added to the C‐terminus, to investigate the effect of N‐terminal acetylation on the conformation and stability of the N‐terminal domain of the coiled‐coil protein. In aqueous buffers at low ionic strength, the reduced, unacetylated 32mer had a very low α‐helical content (approximately 20%) that was only slightly increased by disulfide crosslinking or N‐terminal acetylation. Addition of salt (> 1 M) greatly increased the helical content of the peptide. The CD spectrum, the cooperativity of folding of the peptide, and sedimentation equilibrium ultracentrifugation studies showed that it formed a 2‐chained coiled coil at high ionic strength. Disulfide crosslinking and N‐terminal acetylation both greatly stabilized the coiled‐coil α‐helical conformation in high salt.Addition of ethanol or trifluoroethanol to solutions of the peptide also increased its α‐helical content. However, the CD spectra and unfolding behavior of the peptide showed no evidence of coiled‐coil formation. In the presence of the organic solvents, N‐terminal acetylation had very little effect on the conformation or stability of the peptide.Our results indicate that N‐terminal acetylation stabilizes coiled‐coil formation in the peptide. The effect cannot be explained by interactions with the “helix‐dipole” because the stabilization is observed at very high salt concentrations and is independent of pH. In contrast to the results with the peptide, N‐terminal acetylation has only small effects on the overall stability of tropomyosin.
Mutational and crystallographic analyses of the active site residues of the bacillus circulans xylanase
Authors: Wakarchuk Warren W. , Campbell Robert L. , Sung Wing L. , Davoodi Jamshid , Yaguchi Makoto 
Published: July 2010

AbstractUsing site‐directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme‐substrate complex to 1.8 Å resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid‐base catalyst in the reaction.
MIF proteins are not glutathione transferase homologs
Authors: Pearson William R. 
Published: July 2010

AbstractAlthough macrophage migration inhibitory factor (MIF) proteins conjugate glutathione, sequence analysis does not support their homology to other glutathione transferases. Glutathione transferases are not detected with MIF proteins in searches of protein sequence databases, and MIF proteins do not share significant sequence similarity with glutathione transferases. Homology cannot be demonstrated by multiple sequence alignment or evolutionary tree construction; such methods assume that the proteins being analyzed are homologous.
A proposal for a coherent mammalian histone H1 nomenclature correlated with amino acid sequences
Authors: Parseghian Missag H. , Henschen Agnes H. , Krieglstein Kerstin G. , Hamkalo Barbara A. 
Published: July 2010

AbstractBio‐Rex 70 chromatography was combined with reverse‐phase (RP) HPLC to fractionate histone H1° and 4 histone H1 subtypes from human placental nuclei as previously described (Parseghian MH et al., 1993, Chromosome Res 1:127‐139). After proteolytic digestion of the subtypes with Staphylococcus aureus V8 protease, peptides were fractionated by RP‐HPLC and partially sequenced by Edman degradation in order to correlate them with human spleen subtypes (Ohe Y, Hayashi H, Iwai K, 1986, J Biochem (Tokyo) 100:359‐368; 1989, J Biochem (Tokyo) 106:844‐857). Based on comparisons with the sequence data available from other mammalian species, subtypes were grouped. These groupings were used to construct a coherent nomenclature for mammalian somatic Hls. Homologous subtypes possess characteristic patterns of growth‐related and cAMP‐dependent phosphorylation sites. The groupings defined by amino acid sequence also were used to correlate the elution profiles and electrophoretic mobilities of subtypes derived from different species. Previous attempts at establishing an H1 nomenclature by chromatographic or electrophoretic fractionations has resulted in several misidentifications. We present here, for the first time, a nomenclature for somatic Hls based on amino acid sequences that are analogous to those for H1° and Hlt. The groupings defined should be useful in correlating the many observations regarding H1 subtypes in the literature.
Isoprenyl diphosphate synthases: Protein sequence comparisons, a phylogenetic tree, and predictions of secondary structure
Authors: Chen Anjun , Dale Poulter C. , Kroon Paulos A. 
Published: July 2010

AbstractIsoprenyl diphosphate synthases are ubiquitous enzymes that catalyze the basic chain‐elongation reaction in the isoprene biosynthetic pathway. Pairwise sequence comparisons were made for 6 farnesyl diphosphate synthases, 6 geranylgeranyl diphosphate synthases, and a hexaprenyl diphosphate synthase. Five regions with highly conserved residues, two of which contain aspartate‐rich DDXX(XX)D motifs found in many prenyltransferases, were identified. A consensus secondary structure for the group, consisting mostly of α‐helices, was predicted for the multiply aligned sequences from amino acid compositions, computer assignments of local structure, and hydropathy indices. Progressive sequence alignments suggest that the 13 isoprenyl diphosphate synthases evolved from a common ancestor into 3 distinct clusters. The most distant separation is between yeast hexaprenyl diphosphate synthetase and the other enzymes. Except for the chromoplastic geranylgeranyl diphosphate synthase from Capsicum annuum, the remaining farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families.
Matrix‐assisted laser desorption mass spectrometric peptide mapping of proteins separated by two‐dimensional gel electrophoresis: Determination of phosphorylation in synapsin I
Authors: Zhang Wenzhu , Czernik Andrew J. , Chait Brian T. , Yungwirth Tom , Aebersold Ruedi 
Published: July 2010

AbstractA technique is described for the rapid, sensitive analysis of posttranslational modifications of proteins that have been separated by 2‐dimensional electrophoresis and blotted onto a membrane with a cationic surface. The isolated protein spots visualized by reverse staining of the blotting membrane are excised, washed, and subjected to chemical (cyanogen bromide) and/or enzymatic (endoproteinase Lys‐C) degradation directly on the membrane. The resulting mixture of peptide fragments is extracted from the membrane into a solution that is compatible with matrix‐assisted laser desorption mass spectrometric analysis and analyzed without fractionation. Relatively accurate (± 1 Da) mass determination of these peptide fragments provides a facile and sensitive means for detecting the presence of modifications and for correlating such modifications with the differential mobility of different isoforms of a given protein during 2‐dimensional electrophoresis. The technique is applied to the determination of sites of phosphorylation in synapsins Ia and Ib, neuronal phosphoproteins that are believed to function in the regulation of neurotransmitter release and are substrates for cAMP and Ca2+/calmodulin‐dependent protein kinases, which appear to control their biological activity.
Charge reversal at the P3′ position in protein C optimally enhances thrombin affinity and activation rate
Authors: Richardson Mark A. , Gerlitz Bruce , Grinnell Brian W. 
Published: July 2010

AbstractWe have examined the properties of several human protein C (HPC) derivatives with substitutions for acidic residues near the thrombin cleavage site, including changing the P3′ Asp to Asn (D172N), Gly (D172G), Ala (D172A), or Lys (D172K). The rate of thrombin‐catalyzed activation of D172N, D172G, and D172A was increased 4‐9‐fold compared to wildtype HPC, primarily due to a reduction in the inhibitory effect of calcium and a resulting increase in affinity for free a‐thrombin. There was no significant increase in activation rate or affinity with these 3 derivatives in the absence of calcium, confirming that P3′ Asp affects calcium dependency in the native protein C molecule. With charge reversal at P3′ (D172K), there was a 30‐fold increase in activation rate in the presence of calcium, but unlike the other derivatives, there was a substantial effect (5‐fold) on the activation rate and affinity for free α‐thrombin in the absence of calcium. Thus, protein C affinity for thrombin appears to be influenced by a combination of calcium‐dependent and ‐independent effects of the acidic P3′ residue.
Contribution of a single heavy chain residue to specificity of an anti‐digoxin monoclonal antibody
Authors: Schildbach Joel F. , Shaw Shyh‐Yu , Panka David J. , Near Richard I. , Bruccoleri Robert E. , Novotny Jiri , Sheriff Steven , Jeffrey Philip D. , Haber Edgar , Herzenberg Leonard A. , Jager Gina C. , Parks David R. , Margolies Michael N. 
Published: July 2010

AbstractTwo distinct spontaneous variants of the murine anti‐digoxin hybridoma 26‐10 were isolated by fluorescenceactivated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26‐10. Replacement of Asn with Gln reduced affinity for digoxin 10‐fold relative to the wild‐type antibody, but maintained wild‐type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90‐fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16‐acetylated haptens and haptens with a saturated lactone.The X‐ray crystal structure of the 26‐10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acud Sci USA 90: 10310‐10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild‐type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild‐type and Gln‐mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild‐type hydrogen bonding, and molecular modeling suggests that the reduced side‐chain volume also provides space that can accommodate a congener with a 16‐acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.
Comparison of the structures and the crystal contacts of trypanosomal triosephosphate isomerase in four different crystal forms
Authors: Radhakishan K.V. , Zeelen Johan P.H. , Noble Martin E.M. , Borchert Torben V. , Wierenga Rik K. 
Published: July 2010

AbstractTriosephosphate isomerase (TIM) is a dimeric enzyme consisting of 2 identical subunits. Trypanosomal TIM can be crystallized in 4 different spacegroups: P212121, C2(big cell), C2(small cell), and P1. The P1 crystal form only grows in the presence of 1.4 M DMSO; there are 2 DMSO binding sites per subunit. The structures have been refined at a resolution of 1.83 Å, 2.10 Å, 2.13 Å, and 1.80 Å, respectively. In the 4 different spacegroups the TIM subunit can be observed in the context of 7 different crystallographic environments. In the C2 cells, the dimer 2‐fold axis coincides with a crystallographic 2‐fold axis. The similarities and differences of the 7 subunits are discussed. In 6 subunits the flexible loop (loop 6) is open, whereas in the P212121 cell, the flexible loop of subunit 2 is in an almost closed conformation. The crystal contacts in the 4 different crystal forms are predominantly generated by polar residues in loops. A statistical analysis of the residues involved in crystal contacts shows that, in particular, serines are frequently involved in these interactions; 19% of the exposed serines are involved in crystal contacts.
Crystal structures of pheasant and guinea fowl egg‐white lysozymes
Authors: Lescar Julien , Souchon Hélene , Alzari Pedro M. 
Published: July 2010

AbstractThe crystal structures of pheasant and guinea fowl lysozymes have been determined by X‐ray diffraction methods. Guinea fowl lysozyme crystallizes in space group P6122 with cell dimensionsa= 89.2 Å andc= 61.7 Å. The structure was refined to a final crystallographicR‐factor of 17.0% for 8,854 observed reflections in the resolution range 6‐1.9 Å. Crystals of pheasant lysozyme are tetragonal, space group P43212, witha= 98.9 Å,c= 69.3 Å and 2 molecules in the asymmetric unit. The finalR‐factor is 17.8% to 2.1 Å resolution. The RMS deviation from ideality is 0.010 Å for bond lengths and 2.5° for bond angles in both models. Three amino acid positions beneath the active site are occupied by Thr 40, Ile 55, and Ser 91 in hen, pheasant, and other avian lysozymes, and by Ser 40, Val 55, and Thr 91 in guinea fowl and American quail lysozymes. In spite of their internal location, the structural changes associated with these substitutions are small. The pheasant enzyme has an additional N‐terminal glycine residue, probably resulting from an evolutionary shift in the site of cleavage of prelysozyme. In the 3‐dimensional structure, this amino acid partially fills a cleft on the surface of the molecule, close to the Caatom of Gly 41 and absent in lysozymes from other species (which have a large side‐chain residue at position 41: Gln, His, Arg, or Lys). The overall structures are similar to those of other C‐type lysozymes, with the largest deviations occurring in surface loops. Comparison of the unliganded and antibody‐bound models of pheasant lysozyme suggests that surface complementarity of contacting surfaces in the antigen‐antibody complex is the result of local, small rearrangements in the epitope. Structural evidence based upon this and other complexes supports the notion that antigenic variation inc‐type lysozymes is primarily the result of amino acid substitutions, not of gross structural changes.
Thermodynamic characterization of an artificially designed amphiphilic α‐helical peptide containing periodic prolines: Observations of high thermal stability and cold denaturation
Authors: Kitakuni Eiichi , Kuroda Yutaka , Oobatake Motohisa , Tanaka Toshiki , Nakamura Haruki 
Published: July 2010

AbstractTo investigate the structural stability of proteins, we analyzed the thermodynamics of an artificially designed 30‐residue peptide. The designed peptide, NH2‐EELLPLAEALAPLLEALLPLAEALAPLLKK‐COOH (PERI COIL‐l), with prolines at i + 7 positions, forms a pentameric α‐helical structure in aqueous solution. The thermal denaturation curves of the CD at 222 nm (pH 7.5) show an unusual cold denaturation occurring well above 0 °C and no thermal denaturation is observable under 90 °C. This conformational change is reversible and depends on peptide concentration. A 2‐state model between the monomeric denatured state (5D) and the pentameric helical state (H5) was sufficient to analyze 5 thermal denaturation curves of PERI COIL‐1 with concentrations between 23 and 286 μM. The analysis was carried out by a nonlinear least‐squares method using 3 fitting parameters: the midpoint temperature, Tm, the enthalpy change, ΔH(Tm), and the heat capacity change, Δ Cp. The association number (n= 5) was determined by sedimentation equilibrium and was not used as a fitting parameter. The heat capacity change suggests that the hydrophobic residues are buried in the helical state and exposed in the denatured one, as it occurs normally for natural globular proteins. On the other hand, the enthalpy and the entropy changes have values close to those found for coiled‐coils and are quite distinct from typical values reported for natural globular proteins. In particular, the enthalpy change extrapolated at 110 °C is about 3 kJ/mol per amino acid residue, i.e., half of the value found for globular proteins. Thus, the helices of PERI COIL‐1, observed by CD, would be stabilized by entropic effect rather than enthalpic effect. This might be a general feature for de novo designed proteins that lack the rigid tertiary structure, and are mainly stabilized by nonspecific hydrophobic interactions, as well as for some molten globules of natural proteins.
Helix propensities of the amino acids measured in alanine‐based peptides without helix‐stabilizing side‐chain interactions
Authors: Chakrabartty Avijit , Kortemme Tanja , Baldwin Robert L. 
Published: July 2010

AbstractHelix propensities of the amino acids have been measured in alanine‐based peptides in the absence of helix‐stabilizing side‐chain interactions. Fifty‐eight peptides have been studied. A modified form of the Lifson‐Roig theory for the helix‐coil transition, which includes helix capping (Doig AJ, Chakrabartty A, Klingler TM, Baldwin RL, 1994, Biochemistry 33:3396‐3403), was used to analyze the results. Substitutions were made at various positions of homologous helical peptides. Helix‐capping interactions were found to contribute to helix stability, even when the substitution site was not at the end of the peptide. Analysis of our data with the original Lifson‐Roig theory, which neglects capping effects, does not produce as good a fit to the experimental data as does analysis with the modified Lifson‐Roig theory. At 0 °C, Ala is a strong helix former, Leu and Arg are helix‐indifferent, and all other amino acids are helix breakers of varying severity. Because Ala has a small side chain that cannot interact significantly with other side chains, helix formation by Ala is stabilized predominantly by the backbone (“peptide H‐bonds”). The implication for protein folding is that formation of peptide H‐bonds can largely offset the unfavorable entropy change caused by fixing the peptide backbone. The helix propensities of most amino acids oppose folding; consequently, the majority of isolated helices derived from proteins are unstable, unless specific side‐chain interactions stabilize them.
Structural characteristics and stabilizing principles of bent β‐strands in protein tertiary architectures
Authors: Daffner Carola , Chelvanayagam Gareth , Argos Patrick 
Published: July 2010

Abstractβ‐Strands as constituents of β‐pleated sheets in protein tertiary structures often display considerable distortion from a purely extended conformation. The dislocation types are often characterized as “bulging,” “twisting,” and “bending.” The former 2 properties have been extensively studied and classified. In this work an investigation of bent β‐structures is undertaken. The structural characteristics examined included the bending angles within and out of the principal strand plane, their distribution among various strand types such as parallel and antiparallel, the amino acid preferences at bend sites, and the usage of charged and polar residues for stabilization through interactive anchoring with other atoms of the β‐sheet within which the bent strand lies.