摘要:

AbstractThe structure of a complex of staphylococcal nuclease with Ca2+and deoxythymidine 3′,5′‐biophosphate (pdTp) has been refined by stereochemically restrained leastsquares minimization to a crystallographicRvalue of 0.161 at Å resolution. The estimated root‐mean‐square (rms) error in the coordinates in 0.16 Å. The final model comprises 1082 protein atoms, onecalcium ion, the pdTp molecule, and 82 protein atoms, onecalcium ion, the pdTp molecule, and 82 solvent water molecules;it displays an rms deviation from ideality of 0.017 Å for bond distances and 1.8° for bond angles.The mean distance between corresponding α carbons in the refined and unrefined structures is 0.6 Å we observe small but significant differences between the refined and unrefined models in the turn between residues 27 and 30, the loop between residues 44 and 50, the first helix, and the extended strand between residues 112 and 117 which forms part of the active site binding pocket.The details of the calcium liganding and solvent structure in the activesite are clearly shown in the final electron density map. The structure ofthe catalytic site is consistent with mechanism that has been proposed for this enzyme. However, we note that two lysines from a symmetry‐related molecule in the crystal lattice may play an important role in determining the geometry of inhibitor binding, and that only one of the two required calcium ions is observed in the crystal structure; thus, caution is advised in extrapolating from the structure of the complex of enzyme and inhibitor to that enz

ISSN:0887-3585    

DOI:10.1002/prot.340050302

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

年代:1989

数据来源: WILEY

摘要:

AbstractA mutant Cro protein, which bears the Ile‐30→ Leu substitution, is thermally unstable and degraded more rapidly than wildtype Cro in vivo. Using an antibody screen, we have isolated five different second site suppressor substitutions that reduce the proteolytic hypersensitivity of this mutant Cro protein. Two of the suppressor substitutions increase the thermal stability of Cro by 12°C to 14°C. These amino acid substitutions affect residues 16 and 26, which are substitutions affect residues 16 and 26, which are substantially exposed to solvent in the crystal structure of wild‐t

ISSN:0887-3585    

DOI:10.1002/prot.340050303

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

年代:1989

数据来源: WILEY

摘要:

AbstractCD spectra in the aromatic region of a series of the mutant α‐subunits of tryptophan synthase fromEscherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25°C. The measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild‐type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild‐type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of thewild‐type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild‐type proteins. These residues are polar. In the third group, the CDvalues of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild‐type protein, except for oneband at 276.5 nm. these results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residuesa

ISSN:0887-3585    

DOI:10.1002/prot.340050304

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe function of the N‐terminal amino acids ofSaccharomyces cerevisiaehexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form).This short form of hexokinase II was produced from a fusion between the promoter region of thePGK1gene and theHXK2coding sequence except the first 15 codons. As expected, the in vitro analysis of the short from protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild‐type protein dimerizes. Kinetic studies show that the enzymatic activities are very similarto wild‐type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form ofthe enzyme is similar to the wild‐type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N‐terminalamino acids of hexokinase II are not required in vivo either for phosporylation of hexoses or for glucose r

ISSN:0887-3585    

DOI:10.1002/prot.340050305

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

年代:1989

数据来源: WILEY

摘要:

AbstractIn the studies of the size and structure of multienzyme complexes, a procedure complementary to electron microscopy for determining the molecular dimensions of hydrated multisubunit complexes is needed. For some applications this procedure must be capable of detecting aggregation of complexes and must be applicable to impure preparations. In the present study, a procedure of two‐dimensional agarose gel electrophoresis (2d‐AGE) (Serwer, P. et al. Anal. Biochem. 152: 339–345, 1986) was modified and employed to provide accurate sizemeasurements of several classical multienzyme complexes. To improve band clarity and to achieve required gel pore sizes, a hydroxyethylated agarose was used. The effective pore's radius (PE) as a function of gel concentration was determined for this agarose inthe range ofPEvalue needed for multienzyme complexes (effective radius, R = 10–30 nm). Appropriate conditions wereestablished to measureRvalue ± 1% of the pyruvate (PDC), α‐ketoglutarate (α‐KGDC), and the branched chain α‐keto acid (BCDC) dehydrogenase multienzyme complexes; the accuracy ofRwas limited by the accuracy of the determinations of theRvalue for the sizestandards. The PDC from bovine heart was found to have anR= 22.4 ± 0.2 nm following cross‐linking with glutaraldehyde that was necessary for stabilization of the complex. Dimers and trimers of PDC, present in the preparations used, were separated from monomeric PDCduring 2d‐AGE. AllRvalues for the enzyme complexes studied were agreement with, though more accurate than,Rvaluesobtained by use of electron microscopy. In contrast to this statement, the internal dihydrolipoyl transacetylase core of PDC (E2) had anRof 18.8 ± 0.2 nm using 2d‐AGE, but 10.5 nm by electron microscopy. This observation confirms the proposal that the core of the PDC has externally projecting fibrous domains invisi

ISSN:0887-3585    

DOI:10.1002/prot.340050306

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

年代:1989

数据来源: WILEY

摘要:

AbstractPKB is a computer program system that combines a data base of three‐dimensional protein structures with a series of algorithms for pattern recognition, data analysis, and graphics. By typing relatively simple commands the user may search the data base for instances of a structural motif and analyze in detail the set of individual structures that are found. The application of PKB to the study of protein folding is illustrated in three examples. The first analysis compares the conformations observed for a short sequential motif, sequences similar to the cell‐attachment signal Arg‐Gly‐Asp. The second compares sequences observed for a conformational motif, a 16‐residue βαβ unit. The third analysis considers a population of substructures containing ion‐pair interaction, examining the relationship offrequency of occurrence to calculated electr

ISSN:0887-3585    

DOI:10.1002/prot.340050307

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe role of the electrostatic interactions in the stability of the icosahedral β60capsid of heavy riboflavin synthase fromBacillus Subtilishas been investigated using an approach based on the theory of Kirkwood and Tanford. The pH dependence of the electrostatic subunit interaction agrees well with experimental data. The electrostatic subunit interaction energy has a pronounced minimum at pH 8.2 for both the ligated and ligand‐free capsid. The latter is characterized by a reduction of the magnitude and the pH range of the electrostatic attraction. It is found that only 8 charged groups, which form one cluster and two ion pairs, provide a significant contribution to the capsid stability. The analysis has shown that the aggregation/disaggregation equilibrium seems to be regulated by electrostatic interactions between β‐subunits forming dimers, which connect the relatively stable pentamers in the β‐60capsid. The release of the ligand causesareduction of the electrostatic attraction of the dimers, which may induce disaggregation of the capsid. The electrostatic potential field due tothe titratable groups and α‐helix macrodipoles has been calculated on the basic of the Coulomb relation. Two different values of the dielectric constant have been used for the protein and the surrounding solvent, respectively. The electrostatic potential shows a radially polardistribution with a positive pole at the inner capsid wall and a negative pole outside the capsid. An interesting feature of the electrostatic field is the formation of the positive potential “channels” that coincide with the channels constituted by the pentameric and trimeric β‐subunit aggregates. It is supposed that the electrostatic potential field plays a role in enzyme‐s

ISSN:0887-3585    

DOI:10.1002/prot.340050308

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

年代:1989

数据来源: WILEY

ISSN:0887-3585    

DOI:10.1002/prot.340050301

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

年代:1989

数据来源: WILEY