ISSN:0887-3585    

DOI:10.1002/prot.340060302

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.340060303

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe functions of G proteins—like those of bacterial elongation factor (EF) Tu and the 21 kDa ras proteins (p21ras)—depend upon their abilities to bind and hydrolyze GTP and to assume different conformations in GTP‐ and GDP‐bound states. Similarities in function and amino acid sequence indicate that EF‐Tu, p21ras, and G protein α‐chains evolved from a primordial GTP‐binding protein. Proteins in all three families appear to share common mechanisms for GTP‐dependent conformational change and hydrolysis of bound GTP. Biochemical and molecular genetic studies of the α‐chain of Gs(αs) point to key regions that are involved in GTP‐dependent conformational change and in hydrolysis of GTP. Tumorigenic mutations of αsin human pituitary tumors inhibit‐the protein's GTPase activity and cause constitutive elevation of adenylyl cyclase activity. One such mutation replaces a Gln residue in αsthat corresponds to Gln‐61 of p21ras; mutational replacements of this residue in both proteins inhibit their GTPase activities. A second class of the GTPase inhibiting mutations in αsoccurs in the codon for an ARG residue whose covalent modification by cholera toxin also inhibits GTP hydrolysis by αs. This Arg residue is located in a domain of αsnot represented in EF‐Tu or p21ras. We propose that this domain constitutes an intrinsic activator of GTP hydrolysis, and that it performs a function analogous to that performed for EF‐Tu by the programmed ribosome and for p21rasby the recently discovered GTPase‐activating protein. Owing to their inherited similarities of structure and function, what we learn about αs, p21ras, or EF‐tu as individual molecules helps us to understand crucial functi

ISSN:0887-3585    

DOI:10.1002/prot.340060304

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

年代:1989

数据来源: WILEY

摘要:

AbstractTyrosine 319 ofE. colitopoisomerase I is shown to be the activesite tyrosine that becomes covalently attached to a DNA 5′ phosphoryl group during the transient breakage of a DNA internucleotide bond by the enzyme. The tyrosine was mapped by trapping the covalent complex between the DNA and DNA topoisomerase I, digesting the complex exhaustively with trypsin, and sequencing the DNA‐linked tryptic peptide. Site‐directed mutagenesis converting Tyr‐319 to a serine or phenylalanine completely inactivates the enzyme. The structure of the enzyme andits catalysis of DNA strand breakage, passage, and rejoining are discussed in terms of the available info

ISSN:0887-3585    

DOI:10.1002/prot.340060305

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

年代:1989

数据来源: WILEY

摘要:

AbstractA combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN′, which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wildtype subtilisin BPN′ is greatly restricted by substitution of the catalytic histidine 64 with alanine (H64A) so that certain histidine‐containing substrates are preferentially hydrolysed (Carter, P., Wells, J. A. Science 237:394–399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20‐fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild‐type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain ofStaphylococcus aureusprotein A andEscherichia colialkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N‐terminal sequence. Several features of H64A variants of subtilisin make them attractive for site‐specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N‐terminal side of the cleavage site and yet are broadly specific on the C‐terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized

ISSN:0887-3585    

DOI:10.1002/prot.340060306

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

年代:1989

数据来源: WILEY

摘要:

AbstractLimited digestion ofE. coliDNA topoisomerase I with trypsin or papain generated a DNA‐binding domain of MW 14,000 corresponding to the carboxyl terminal of the enzyme. This fragment binds to single‐stranded DNA agarose as tightly as the intact enzyme. It required around 400 mM NaCl for elution. A truncated topoisomerase that lacks this C‐terminal domain was purified. It was eluted from the single‐stranded DNA agarose column at around 150 mM NaCl. Although the truncated enzyme could relax negatively supercoiled DNA as efficiently as the intact enzyme at low ionic strength, its processivity was more sensitive to increasing salt concentration. Measurement of binding to fluorescent etheno‐M13 DNA also demonstrated that the presence of the C‐terminal domain confers higher affinity to DNA for

ISSN:0887-3585    

DOI:10.1002/prot.340060307

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe rapid kinetic phase that leads from unfolded species to transient folding intermediates in dihydrofolate reductase fromEscherichia coliwas examined by site‐directed mutagenesis and by physicochemical means. The absence of this fluorescence‐detected phase in the refolding of the Trp‐74Phe mutant protein strongly implies that this early phase in refolding can be assigned to just one of the five Trp residues in the protein, Trp‐74. In addition, water‐soluble fluorescence quenching agents, iodide and cesium, have a much less significant effect on this early step in refolding than on the slower phases that lead to native and nativelike conformers. These and other data imply that an important early event in the folding of dihydrofolate reductase is the formation of a hydrophobic cluster which protects Trp‐74

ISSN:0887-3585    

DOI:10.1002/prot.340060308

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

年代:1989

数据来源: WILEY

摘要:

AbstractPharmacological analysis of ligand binding to the β‐adrenergic receptor (βAR) has revealed the existence of two distinct receptor subtypes (β1and β2) which are the products of different genes. The predicted amino acid sequence of the β1and β2receptors differ by 48%. To identify the regions of the proteins responsible for determining receptor subtype, chimeras were constructed from domains of the human β1and hamster β2receptors. Analyses of the ligand‐binding characteristics of these hybrid receptors revealed that residues in the middle portion of the βAR sequence, particularly around transmembrane regions 4 and 5, contribute to the subtype specific binding of agonists. Smaller molecular replacement of regions of the hamster β2AR with the analogous regions from the avian β1AR, however, failed to identify any single residue substitution capable of altering the subtype specificity of the receptor. These data indicate that, whereas sequences around transmembrane regions 4 and 5 may contribute to conformations which influence the ligand‐binding properties of the receptor, the subtype‐specific differences in amine‐substituted agonist binding cannot be attributed to a single molecular interaction between the ligand and any amino acid residue which is divergent between t

ISSN:0887-3585    

DOI:10.1002/prot.340060309

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

年代:1989

数据来源: WILEY

摘要:

AbstractSite saturation mutagenesis has been carried out at Ala‐237 in RTEM‐1 β‐lactamase to assess the role of this site in modulating differences in specificity of β‐lactamases for penams vs. cephams as substrates. (An Ala‐237 Thr mutation had previously been shown to increase activity on cephems by about 30–80%.1,2) Screening of all 19 possibles mutants on penams and cephems revealed the even more active Ala‐237 Asn mutant. Detailed kinnetic analysis showns that this mutant has about four times the activity toward cephalothin and cephalosporin C as the wild‐type enzyme. Both mutations reduce the activity toward penams to about 10% that of RETM‐1 β‐lactamase and lower by about 5°C the tempreature at which the enzyme denatures. Functional properties of the other mutants have also been surveyed. The most intresting aspect of these results is that two quite disparate amino acids, theronine and asparagine, when intorduced for Ala‐237, cause such similar changes in enzyme specificity while more similar residues do not alter the catalytic properties of the enzyme to

ISSN:0887-3585    

DOI:10.1002/prot.340060310

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

年代:1989

数据来源: WILEY

摘要:

AbstractCalmodulin is known to bind target enzymes and basic, amphiphilic peptides in a Ca2+‐dependent manner. Recently, we introduced a photoaffinity label,p‐benzoylphenylalanine (Bpa), into the sequence of a model, α‐helical, calmodulin‐binding peptide. When the Bpa residue was introduced at the third position of the peptide, Met‐144 on the C‐terminal domain of calmodulin was labeled, whereas when the photolabel was placed at the thirteenth position, Met‐71 on the N‐terminal do main was labeled. Assuming that both peptides bind in similar orientations, these results are not consistent with the crystal structure of calmodulin, in which the domains are held at a significant distance from one another by a long α‐helical segment. To test the assumption that both peptides bind in similar orientations, we have synthesized a calmodulin‐binding peptide with the photolabel in both the third and the thirteenth positions. Upon photolysis, this peptide forms a cross‐link between Met‐71 and Met 124 on the N‐ and C‐terminal domains, respectively. Furthermore, a peptide with a Bpa in the thirteenth position and a Trp residue in the third position was also synthsized. After photocross‐linking the Bpa redidue of this peptide to Met‐71 of calmodulin, it could be shown that the fluorescence properties of the Trp residue were consistent with its side chain being buried in a hydrophobic pocket on the C‐terminal domain of calmodulin. These data indicate that, when complexed with basic, amphiphi peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal st

ISSN:0887-3585    

DOI:10.1002/prot.340060311

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

年代:1989

数据来源: WILEY