bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural analysis of purified beta‐adrenergic receptors
Authors: Fraser Claire M. , Kerlavage Anthony R. , Mariani Andrew P. , Venter J. Craig 
Published: May 2005

AbstractWe have characterized the structure of purified beta‐adrenergic receptors by a combination of photoaffinity labeling, high performance liquid chromatography (HPLC)‐tryptic mapping, CNBr fragmentation, target size analysis, and electron microscopy of purified receptor molecules. Guinea pig lung beta‐adrenergic receptors purified by affinity chromatography, ion exchange chromatography, and HPLC size exclusion chromatography or photoaffinity labeled with [125]‐iodocyanopindolol diazirine displayed mobilities on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) that corresponded to Mr = 68,000. Purified, radioiodinated guinea pig lung beta‐receptors were subjected to complete trypsin digestion and subsequent reverse‐phase HPLC analysis, which revealed nine peptides. Active site labeling and tryptic digestion of partially purified hamster lung beta‐receptors produced one peptide, whereas CNBr digestion of the same material produced two labeled fragments, yielding information about the location of the active site within the primary sequence. Purified guinea pig lung receptors were examined with transmission electron microscopy. Electron micrographs revealed slightly asymmetric, rod‐shaped structures with an average length of 13 nm and width of 3.4 nm. Many receptors were arranged as apparent dimeric structures. These findings confirm data obtained from target size analysis of guinea pig lung beta‐receptors in situ which suggest that receptors may exist as oligomeric arrays in the native membrane. Taken together, these data provide information about putative functional domains of the beta‐adrenergic receptor and its quaternary structure.
Distribution of accessible surfaces of amino acids in globular proteins
Authors: Lawrence Charles , Auger Ivan , Mannella Carmen 
Published: May 2005

AbstractGlobular proteins fold into compact particles with interior amino acid residues shielded from the surounding aqueous environment. An early hypothesis holds that entropic hydophobic forces dirve this phenomenon. However, previous analyses based on a binary description of the accesible surfaces of amino acid residues in proteins did not support theis hypothesis. This report shows that a complete description of accessible surface areas is given by parametric distribution fuctions with three modes. The modes are formed by partitioning the available accessible surface area of the amino acids into three segments; the data for each segment are characterized by a mode‐specific model. In the “repulsive” mode, probabilities of accessibility decrease exponentially with exposed surface area, as predicted by the hydrophobic hypothesis. A distinct “buried” mode is needed to account for an excess of residues at or near zero accessibility for most amino acids, consistent with the use of binary descriptions of accessibility. A third mode exists which is termed “near neutral” because it is described by a nearly uniform distribution of accessibility for the hydrophiliic amino acids. Empirical energies calculated for the repulsive mode correlate well with measured free energies of transfer of amino acids from water to organic solvents, while those from the buried mode correlate well with measured free energies of hydration of the side chains. Poor cross correlations between these energies give an explanation for the previous conflict in interpreting these data.
Structure of an insect virus at 3.0 Å resolution
Authors: Hosur M.V. , Schmidt T. , Tucker R.C. , Johnson J.E. , Gallagher T.M. , Selling B.H. , Rueckert R.R. 
Published: May 2005

AbstractWe report the first atomic resolution structure of an insect virus determined by single crystal X‐ray diffraction. Black beetle virus has a bipartite RNA genome encapsulated in a single particle. The capsid contains 180 protomers arranged on a T = 3 surface lattice. The quaternary organization of the protomers is similar to that observed in the T = 3 plant virus structures. The protomers consist of a basic, crystallographically disordered amino terminus (64 residues), a β‐barrel as seen in other animal and plant virus subunits, an outer protrusion composed predominantly of β‐sheet and formed by three large insertions between strands of the barrel, and a carboxy terminal domain composed of two distorted helices lying inside the shell. The outer surfaces of quasi‐threefold related protomers form trigonal pyramidyl protrusions. A cleavage site, located 44 residues from the carboxy terminus, lies within the central cavity of the protein shell.The structural motif observed in BBV (a shell composed of 180 eight‐stranded antiparallel β‐barrels) is common to all nonstatellite spherical viruses whose structures have so far been solved. This highly conserved shell architecture suggests a common origin for the coat protein of spherical viruses, while the primitive genome structure of BBV suggests that this insect virus represents an early stage in the evolution of spherical viruses from cellular genes.
Anisotropy and anharmonicity of atomic fluctuations in proteins: Analysis of a molecular dynamics simulation
Authors: Ichiye Toshiko , Karplus Martin 
Published: May 2005

AbstractPositional probability density functions (pdf) for the atomic fluctuations are determined from a molecular dynamics simulation for hen egg‐white lysozyme. Most atoms are found to have motions that are highly anisotropic but only slightly anharmonic. The largest deviations from harmonic motion are in the direction of the largest rms fluctuations in the local principal axis frame. Backbone atoms tend to be more nearly harmonic than sidechain atoms. The atoms with the largest anharmonicities tend to have pdfs with multiple peaks, each of which is close to harmonic. Several model pdfs are evaluated on the basis of how well they fit probability densities from the dynamics simulations when parameterized in terms of the moments of the distribution. Gram‐Charlier and Edgeworth perturbation expansions, which have been successful in describing the motions of small molecules in crystals, are shown to be inadequate for the distributions found in the dynamics of proteins. Multipeaked distribution functions are found to be more appropriate.
Human myeloperoxidase and thyroid peroxidase, two enzymes with separate and distinct physiological functions, are evolutionarily related members of the same gene family
Authors: Kimura Shioko , Ikeda‐Saito Masao 
Published: May 2005

AbstractHuman myeloperoxidase and human thyroid peroxidase nucleotide and amino acid sequences were compared. The global similarities of the nucleotide and amino acid sequences are 46% and 44%, respectively. These similarities are most evident within the coding sequence, especially that encoding the myeloperoxidase functional subunits. These results clearly indicate that myeloperoxidase and thyroid peroxidase are members of the same gene family and diverged from a common ancestral gene. The residues at 416 in myeloperoxidase and 407 in thyroid peroxidase were estimated as possible candidates for the proximal histidine residues that link to the iron centers of the enzymes. The primary structures around these histidine residues were compared with those of other known peroxidases. The similarity in this region between the two animal peroxidases (amino acid 396–418 in thyroid peroxidase and 405–427 in myeloperoxidase) is 74%; however, those between the animal peroxidases and other yeast and plant peroxidases are not significantly high, although several conserved features have been observed. The possible location of the distal histidine residues in myeloperoxidase and thyroid peroxidase amino acid sequences are also discussed.
Peptide synthesis catalyzed by polyethylene glycol‐modified chymotrypsin in organic solvents
Authors: Gaertner Hubert F. , Puigserver Antoine J. 
Published: May 2005

AbstractChymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene‐soluble modified enzyme readily catalyzed both aminolysis of N‐benzoyl‐L‐tyrosine p‐nitroanilide and synthesis of N‐benzoyl‐L‐tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L‐ as well as D‐amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivativesThe acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl‐enzyme with subsequent synthesis of a peptide bond.
Mapping the enzymatic active site of Pseudomonas aeruginosa exotoxin A
Authors: Brandhuber Barbara J. , Allured Viloya S. , Falbel Tanya G. , McKay David B. 
Published: May 2005

AbstractPseudomonas aeruginosa exotoxin A is representative of a class of enzymes, the monoADP‐ribosyl, which catalyze the covalent transfer of an ADP‐ribose moiety of NAD+ to a target substrate. Availability of the three‐dimensional structure of exotoxin A provides the opportunity for mapping substrate binding sites and suggesting which amino acid residues may be involved in catalysis. Data from several sources have been combined to develop a proposal for the NAD+ binding site of exotoxin A: the binding of NAD+ fragments adenosine, AMP, and ADP have been delineated crystallographically to 6.0, 6.0, and 2.7 Å, respectively; significant sequence homology spanning 60 residues has been found between exotoxin A and diphtheria toxin, which has the identical enzymatic activity; iodination of exotoxin A, under conditions in which only tyrosine 481 is iodinated in the enzymatic domain, abolishes ADP‐ribosyl transferase activity.
Crystallization and preliminary X‐ray diffraction study of a protein with a high potential rubredoxin center and a hemerythrin‐type Fe center
Authors: Sieker L. C. , Turley S. , Prickril B. C. , LeGall J. 
Published: May 2005

AbstractA newly discovered iron‐containing protein, isolated from the bacterium Desulfovibrio vulgaris (Hildenborough, NCIB 8303), has been crystallized. The molecule appears to be a dimer of mass 44kDa. This protein has iron centers with spectroscopic similarities to those in rubredoxins and in hemerythrins.The X‐ray diffraction shows symmetry consistent with space group I222 or I212121. Cell parameters are a = 49.2 Å, b = 81.3 Å, c= 100.1 Å, and α, β, γ = 90°. X‐ray diffraction data have been collected to 3.0 Å, and a search for useful heavy atom derivatives is in progress for the analysis of the crystal structure of this Fe‐protein.
Mechanism of protein folding: I. General considerations and refolding of myoglobin
Authors: Saitô Nobuhiko , Shigaki Takao , Kobayashi Yutaka , Yamamoto Masahiko 
Published: May 2005

AbstractTo explain the rapidity of the process of protein folding, we cite two aspects of hydrophobic interaction: its long‐range nature and the specificity of pairing after the formation of secondary structures. These two factors, when incorporated with the growth‐type mechanism, can determine the folding pathway of proteins. This mechanism is applied to myoglobin. Appropriate introduction of side chins of amino acid residues and the heme group attached to His 93 yield a refolded tertiary structure that is in good agreement with the native structure.
Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine‐185 protonates and deprotonantes during the photocycle
Authors: Braiman Mark S. , Mogi Tatsushi , Stern Lawrence J. , Hackett Neil R. , Chao Betty H. , Khorana H. Gobind , Rothschild Kenneth J. 
Published: May 2005

AbstractThe techniques of FTIR difference spectroscopy and site‐directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton‐pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr→Phe substitution, difference spectra have been obtained for the bR→K and bR→M photoreactions. Only the Tyr‐185→Phe mutation results in the disappearance of a set of bands that were previously shown to be due to protonation of a tryosinate during the br→K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United states of America 83:347, (1986)]. The Tyr‐185→Phe mutation also eliminates a set of bands in the bR→M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272‐cm−1 peak) are completely unaffected by the other ten Tyr→Phe mutations. Thus, tyrosinate‐185 gains a proton during the bR→K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr‐185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative CNH+ stretch band shifts from 1640 cm−1 in the wild type to 1636 cm−1 in the Tyr‐185→Phe mutant. A model that is consistent with these results is that Tyr‐185 is normally ionized and serves as a counter‐ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr‐185, which raises the pKa of the latter group and results in its protonation.
The aggregation state of spin‐labeled melittin in solution and bound to phospholipid membranes: Evidence that membrane‐bound melittin is monomeric
Authors: Altenbach Christian , Hubbell Wayne L. 
Published: May 2005

AbstractSpin‐labeled derivatives of the bee venom protein, melittin, were obtained by reacting on the average one of the four amino groups of the protein with succinimidyl‐2,2,5,5‐tetramethyl‐3‐pyrroline‐1‐oxyl‐3‐carboxylate All 16 statistically possible reaction products with 0, 1, 2, 3 or 4 spin labels per protein were then separated in a single pass with reversed phase high performance liquid chromatography. With the help of trypsin digestion and diode array detection it was possible to assign the primary structure of all 16 eluting fractions. All fractions with only one spin label per protein were purified for electron paramagnetic resonance measurement. The labeling sites cover different regions of the protein: one is at the N‐terminus, one at lysine‐7, and two are near the C‐terminus at lysine‐21 and lysine‐23, respectively. This set of specifically labeled melittins was used to study the structure and dynamics of melittin in aqueous solutions and when bound to neutral or negatively charged membranes. In aqueous solution a reduction in rotational correlation time and appearance of spin‐spin interaction was observed during salt‐induced transition from a random coil monomer to a mostly α‐helical retramer. Membrane binding to phospholipid bilayers in low or high ionic strength was reflected only in a further decrease in mobility. The absence of any spin interaction in the membrane‐bound state suggests that melittin is monomeric under these conditions. All derivatives were able to detect these structural changes, but melittin labeled at the N‐terminal amino group was especially valuable. Because of postulated intramolecular hydrogen bonding, this label reflects directly the motion of the entire protein or tetramer. Broadening experiments with chromium oxalate show that all labeled sites are at least partially exposed to the aqueous phase when melittin is bound to membranes. This suggests that an α‐helical melittin monomer binds to membranes with its axis parallel to the membrane surface.
Preferred conformational state of the N‐terminus section of a bovine growth hormone fragment (residues 96‐133) in water is an omega loop
Authors: Gooley P. R. , Carter S. A. , Fagerness P. E. , MacKenzie N. E. 
Published: May 2005

AbstractThe solution structure of a 38‐amino‐acid‐residue, biologically active fragment of bovine growth hormone (bGH96–133) was investigated with a combined nuclear magnetic resonance (NMR) and computer modeling approach. With the distance geometry program DISGEO and distance constraints derived from the nuclear Overhauser enhancement (NOE) experiments, it was found that residues Ser‐100 to Tyr‐110 circumscribe an Ω‐loop, a recently categorized feature of nonregular secondary protein structure.
X‐ray and model‐building studies on the specificity of the active site of proteinase K
Authors: Betzel C. , Bellemann M. , Pal G. P. , Bajorath J. , Saenger W. , Wilson K. S. 
Published: May 2005

AbstractProteinase K, the extracellular serine endopeptidase (E.C. 3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy‐Ala‐Phechloromethyl Ketone to the active site of proteinase K was the first determined from a Fourier synthesis based on synchrotron X‐ray diffraction data between 1.8 Å and 5.0 Å resolution. The protein inhibitor complexes was refined by restrained least‐squares minimization with the data between 10.0 and 1.8 Å. The final R factor was 19.1% and the model contained 2,018 protein atoms, 28 inhibitors atoms, 125 water molecules, and two Ca2+ ions. The peptides portion of the inhibitor is bound to the active center of proteinase K by means of a three‐stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.
Structural analysis of the 2.8 Å model of xylose isomerase from Actinoplanes missouriensis
Authors: Rey Felix , Jenkins John , Janin Joël , Lasters Ignace , Alard Philippe , Classens Michel , Matthyssens Gaston , Wodak Shoshana 
Published: May 2005

AbstractThe structure of Xylose isomerase (X.I.) from Actinoplanes missouriensis has been solved to 2.8 Angstroms resolution. Phases were determined from a single Eu3+ derivative and from the noncrystallographic 22 symmetry of the tetrameric molecule. An atomic model was built and subjected to restrained crystallographic refinement. The resulting model is shown to be closely similar to the recently reported X.I.'s structures from three other bacterial sources. Each monomer is found to be composed of an eight‐stranded α/β “T.I.M.” barrel forming an N‐terminal domain of 328 residues followed by a large loop of 66 residues embracing an adjacent subunit. Analysis of intersubunit packing shows that the X.I. tetramer is an assembly of two tight dimers. The β barrel fits a simple hyperboloid model as other T.I.M. barrels do. The active site, identified as the binding site for the inhibitor xylitol, is located at the carboxyl end of the beta strands in the barrel next to a pair binding site for Eu3+ ions, which are assumed to the sites for the divalent ions involved in catalysis. Active sites in the tetramer are oriented towards the interface between dimmers. It is suggested that subunit interfaces might stabilize the active site region and this might explain the oligomeric nature of the other α/β barrel enzymes.
Formation of heterodimers between wild type and mutant trp aporepressor polypeptides of Escherichia coli
Authors: Graddis Thomas J. , Klig Lisa S. , Yanofsky Charles , Oxender Dale L. 
Published: May 2005

AbstractAvailability of the three‐dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that from the tryptophan binding pocket. Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid. Equilibrium dialysis with 14C‐tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants. The results obtained indicate that replacement of theronine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two‐and four‐fold, respectively. Replacement of ariginine 54 by histidine (RH84) or glycine 85 by ariginine (GR85) results in complete loss of tryptophan binding activity. Purified mutant and wild type aporepressors were used in vitro heterodimer studies. The trp repressor of E. coli functions as a stable dimer. A large number of trp repressor mutants prduces defective repressors that are transdominant to the wild type repressor in vivo. The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits. An in vitro assay was developed to detect and measure heterodimer formation. Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels. Aporepressors readily formed heterodimer formation upon treatment at 65°C for 3 minutes. Heterodimer formation was significantly retarded by the presence of the corepressor, L‐tryptophan. Indole‐3‐propionic acid, 5‐methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation. These results indicate that the presence of the indole moiety in the corepressor binding pocket increases the stability of the dimer.