摘要:

AbstractThe uncertainties in the refined parameters for a 1.5‐Å X‐ray structure of carbon‐monoxy (FeII) myoglobin are estimated by combining energy minimization with least‐squares refinement against the X‐ray data. The energy minimization, done without reference to the X‐ray data, provide perturbed structures which are used to restart conventional X‐ray refinement. The resulting refined structures have the same, or better, R‐factor and stereochemical parameters as the original X‐ray structure, but deviate from it by 0.13 Å rms for the backbone atoms and 0.31 Å rms for the sidechain atoms. Atoms interacting with a disordered sidechain, Arg 45 CD3, are observed to have larger positional uncertainties. The uncertainty in the B‐factors, within the isotropic harmonic motion approximation, is estimated to be 15%. The resulting X‐ray structures are more consistent with the energy parame

ISSN:0887-3585    

DOI:10.1002/prot.340020102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractThe feasibility of detecting human somatic structural gene mutations by two dimensional electrophoresis has been investigated. A lymphoblastoid cell line was grown as a mass culture in the presence of ethylnitrosourea, after which cells were regrown as single cell clones. A total of 257 polypeptide spots were analyzed in gels derived from 186 clones. Four structural mutations were detected by visual analysis of the gels. Computer analysis of gels corresponding to the mutant clones was also undertaken. At a spot size threshold of 200 spots to be matched using a computer algorithm, all four mutant polypeptides were detected. These results indicate the usefulness of the two‐dimensional approach for mutagenesis studies at the protein leve

ISSN:0887-3585    

DOI:10.1002/prot.340020103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractA basic, amphiphilic α helix is a structural feature common to a variety of inhibitors of calmodulin and to the calmodulin‐binding domains of myosin light chain kinases. To aid in recognizing this structural feature in sequences of peptides and proteins we have developed a computer algorithm which searches for sequences of appropriate length, hydrophobicity, helical hydrophobic moment, and charge to be considered as potential calmodulin‐binding sequences. Such sequences occurred infrequently in proteins of known crystal structure. This algorithm was used to find the most likely site in the catalytic (γ) subunit of phosphorylase b kinase for interaction with calmodulin (the δ subunit). A peptide corresponding to this site (residues 341–361 of the γ subunit) was synthesized and found to bind calmodulin with approximately an 11 nM dissociation constant. A variant of this peptide in which an aspartic acid at position 7 in its sequence (347 of the γ subunit) was replaced with an asparagin was found to bind calmodulin with approximately a 3 nM dissociatio

ISSN:0887-3585    

DOI:10.1002/prot.340020104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

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 i

ISSN:0887-3585    

DOI:10.1002/prot.340020105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractImmunological cross‐reaction was employed for identification of proteolytic fragments ofE. coliRNA polymerase genered both in vitro and in vivo. Several species of partially denatured but assembled RNA polymerase were isolated, which were composed of fragments of the two large subunits, β and β′, and the two small and intact subunits, α and σ. Comparison of the rate and pathway of proteolytic cleavage in vitro of unassembled subunits, subassemblies, and intact enzymes indicated that the susceptibility of RNA polymerase subunits to proteolytic degradation was dependent on the assembly state.Using this method, degradation in vivo was found for some, but not all, of the amber fragments of β subunit in merodiploid cells carrying both wild‐type and mutantrpoBgenes. Although the RNA polymerase is a metabolically stable component in exponentially growing cells ofE. coli, degradation of the full‐sized subunits was found in two cases, i.e., several temperature‐sensitiveE. colimutants with a defect in the assembly of RNA polymerase and the stationary‐phase cells of a wild‐typeE. coli. The in vivo degradation of RNA polymerase was indicated to be initiated by alteration of th

ISSN:0887-3585    

DOI:10.1002/prot.340020106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractThe equilibria and kinetics of ureainduced unfolding and refolding of the α subunit of tryptophan synthase ofE. colihave been examined for their dependences on viscosity, pH, and temperature in order to investigate the properties of one of the rate‐limiting steps, domain association. A viscosity enhancer, 0.58 M sucrose, was found to slow unfolding and accelerate refolding. This apparently anomolous results was shown to be due to the stabilizting effect of sucrose on the folding reaction. After accounting for this stabilization effect by using linear free‐energy plots, the unfolding and refolding kinetics were found to have a viscosity dependence. A decrease in pH was found to stabilize the domain association reaction by increasing the refolding rate and decreasing the unfolding rate. This effect was accounted for by protonation of a single residue with a pK value of 8.8 in the native state and 7.1 in the intermediate, in which the two domains are not yet associated. The activation energy of unfolding is 4.8 kcal/mol, close to the diffusion limit. The negative activation entropy of unfolding, −47 cal/deg‐mol, which controls this reaction, may result from ordering of solvent about the newly exposed domain interface of the transition state. These results may provide information on the types of noncovalent interactions involved in domain association and improve the ability to interpret the folding of mutants with single amino‐acid substitutions at the

ISSN:0887-3585    

DOI:10.1002/prot.340020107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractA survey of 47 globular proteins was made to determine the probability of occurrence of ion pairs separated by 1, 2, 3, … and 8 residues in the alpha helices. As a control, the probability of occurrence of like charged pairs was also determined. The survey showed that ion pairs of the type i, i±3 and i, i±4 are the most predominant. Such a preference was not observed for like charged pairs. The observed frequency of ion pairs is significantly greater than their expected frequency. The normalized frequencies of occurrence of the ion pairs were also found to increase generally with the helix length. These results indicate that the ion pairs may contribute to the stability of solvent‐exposed alpha helices. Since the stabilization of protein secondary structure, these results may throw light on the mechanism of protein fo

ISSN:0887-3585    

DOI:10.1002/prot.340020108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractA few years ago we reported that histidine (HC3) 146β plays a major role in the pH‐dependent propeties of the R‐state of human hemoglobin, accounting for close to 50% of the R‐state Bohr effect. We have extended these studies by examining the role of arginine 141α, another group known to affect the overall Bohr effect. We have compared the pH dependencies of the rate constants for the dissociation and combination of the fourth carbon monoxide molecule, I4and I′4, respectively, for native hemoglobin A (HbA) and a control reconstututed HbA, and des‐(Arg 141α) HbA, the hemoglobin molecule resulting from the enzymatic removal of the C–terminal arginine of the α‐chain of human Hb. From these kinetic contants the pH dependence of L4, by about 80% between pH 6 and 8, where the aldkaline Bohr effect normally occurs, The sum of the effects of the removal of His 146β and of Arg 141α is grater than 100%. This suggests that at least one of these modifications altrs the contrubutions of other residues

ISSN:0887-3585    

DOI:10.1002/prot.340020109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

ISSN:0887-3585    

DOI:10.1002/prot.340020101

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY