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Journal Issue - Volume 5 Issue 5 (May 1996)

Abstract A strategy that combines limited proteolysis experiments and mass spectrometrie analysis of the fragments generated has been developed to probe protease‐accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64‐residue protein consisting of a β‐sheet scaffold based on the heavy‐chain variable‐domain structure of a mouse...

Abstract The role of β‐turns in dictating the structure of a β‐barrel protein is assessed by probing the tolerance of the central β‐turn of poplar plastocyanin to substitution by arbitrary sequences. Native plastocyanin binds copper and is colored bright blue. However, when the wild‐type Pro47‐Ser48‐Gly49‐Val50 turn sequence is replaced by arbitrary tetrapeptides, the vast majority (92/98 = 94%) of mutant proteins cannot fold into ...

Abstract Thrombin, a trypsin‐like serine protease present in blood, plays a central role in the regulation of thrombosis and hemostasis. A cyclic pentapeptide, cyclotheonamide A (CtA), isolated from sponges of the genus Theonella, inhibits thrombin, trypsin, and certain other serine proteases. Enzyme inhibition data for CtA indicate that it is a moderate inhibitor of α‐thrombin (Ki = 1.0 nM), but substantially more potent toward trypsin (Ki =...

Abstract Prostate‐specific antigen (PSA), produced by prostate cells, provides an excellent serum marker for prostate cancer. It belongs to the human kallikrein family of enzymes, a second prostate‐derived member of which is human glandular kallikrein‐1 (hK2). Active PSA and hK2 are both 237‐residue kallikrein‐like proteases, based on sequence homology. An hK2 model structure based on the serine protease fold is presented and...

Abstract Lactococcus lactis is the only organism known to contain two dihydroorotate dehydrogenases, i.e., the A‐ and B‐forms. In this paper, we report the overproduction, purification, and crystallization of dihydroorotate dehydrogenase A. In solution, the enzyme is bright yellow. It is a dimer of subunits (34 kDa) that contain one molecule of flavin mononucleotide each. The enzyme shows optimal function in the pH range 7.5–9.0. It...

Abstract Although the exact physiological function of uteroglobin is not known, it has been suggested that it may function by inhibiting phospholipase A2. We have found that the uteroglobin fold is embedded in that of the pore‐forming domain of colicin A. Colicin A is an antibiotic protein that kills sensitive Escherichia coli cells by forming a pore in their phospholipid membrane. The RMS deviation between the Cα atoms after the structural...

Abstract L(+)‐lactate dehydrogenase (LDH; E.C.1.1.1.27) from the hyperthermophilic bacterium Thermotoga maritima has been shown to represent the most stable LDH isolated so far (Wrba A, Jaenicke R, Huber R, Stetter KO, 1990, Eur J Biochem 188:195–201). In order to obtain the enzyme in amounts sufficient for physical characterization, and to analyze the molecular basis of its intrinsic stability, the gene was cloned and expressed functionally in Escherichia...

  • Interaction of subtilisins with serpins

  • Tomoko Komiyama, Hanne Grøn, Guy S. Salvesen, Philip A. Pemberton
  • Published in Wiley Interscience on Dec 31, 2008
  • DOI: 10.1002/pro.5560050509 (p 874-882)

Abstract Serpins are well‐characterized inhibitors of the chymotrypsin family serine proteinases. We have investigated the interaction of two serpins with members of the subtilisin family, proteinases that possess a similar catalytic mechanism to the chymotrypsins, but a totally different scaffold. We demonstrate that α1proteinase inhibitor inhibits subtilisin Carlsberg and proteinase K, and α1antichymotrypsin inhibits proteinase K, but not...

Abstract Based on 2D 1H‐1H and 2D and 3D 1H‐15N NMR spectroscopies, complete 1H NMR assignments are reported for zinc‐containing Clostridium pasteurianum rubredoxin (Cp ZnRd). Complete 1H NMR assignments are also reported for a mutated Cp ZnRd, in which residues near the N‐terminus, namely, Met 1, Lys 2, and Pro 15, have been changed to their counterparts, (‐), Ala and Glu, respectively, in rubredoxin from the hyperthermophilic archaeon,...

  • Protein design automation

  • Bassil I. Dahiyat, Stephen L. Mayo
  • Published in Wiley Interscience on Dec 31, 2008
  • DOI: 10.1002/pro.5560050511 (p 895-903)

Abstract We have conceived and implemented a cyclical protein design strategy that couples theory, computation, and experimental testing. The combinatorially large number of possible sequences and the incomplete understanding of the factors that control protein structure are the primary obstacles in protein design. Our protein design automation algorithm objectively predicts protein sequences likely to achieve a desired fold. Using...

Abstract In order to determine environments around four tryptophan residues, located in the N‐terminus, in the kinase and in the phosphatase domains of rat testis Fru 6‐P,2‐kinase:Fru 2,6‐bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site‐directed mutagenesis. The kinetic constants of these mutant enzymes were similar to those of the wild‐type enzyme. The sum of the fluorescence intensities of the...

Abstract Phosphatidylcholine‐specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols. Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein. Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities...

Abstract α‐Lactalbumin is a small, globular protein that is stabilized by four disulfide bonds and contains two structural domains. One of these domains is rich in α‐helix (the α‐domain) and has Cys 6‐Cys 120 and Cys 28‐Cys 111 disulfide bonds. The other domain is rich in β‐sheet (the β‐domain), has Cys 61‐Cys 77 and Cys 73‐Cys 91 disulfide bonds, and includes one calcium binding site. To investigate the interaction between domains,...

Abstract The i + 5 → i hydrogen bonded turn conformation (7r‐turn) with the fifth residue adopting αL conformation is frequently found at the C‐terminus of helices in proteins and hence is speculated to be a “helix termination signal.” An analysis of the occurrence of i + 5 → i hydrogen bonded turn conformation at any general position in proteins (not specifically at the helix C‐terminus), using coordinates of 228 protein crystal structures...

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