Protein engineering最新文献_第9页

Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo. 一种新的组氨酸类似物的合成及其在体内有效地结合到蛋白质中。

Protein engineering Pub Date : 2003-09-01 DOI: 10.1093/protein/gzg084

Yutaka Ikeda, Shun-ichi Kawahara, Masumi Taki, Atsushi Kuno, Tsunemi Hasegawa, Kazunari Taira

引用次数: 37

PROSPECT II: protein structure prediction program for genome-scale applications. 展望II:基因组级应用的蛋白质结构预测程序。

Protein engineering Pub Date : 2003-09-01 DOI: 10.1093/protein/gzg081

Dongsup Kim, Dong Xu, Jun-tao Guo, Kyle Ellrott, Ying Xu

{"title":"PROSPECT II: protein structure prediction program for genome-scale applications.","authors":"Dongsup Kim, Dong Xu, Jun-tao Guo, Kyle Ellrott, Ying Xu","doi":"10.1093/protein/gzg081","DOIUrl":"https://doi.org/10.1093/protein/gzg081","url":null,"abstract":"A new method for fold recognition is developed and added to the general protein structure prediction package PROSPECT (http://compbio.ornl.gov/PROSPECT/). The new method (PROSPECT II) has four key features. (i) We have developed an efficient way to utilize the evolutionary information for evaluating the threading potentials including singleton and pairwise energies. (ii) We have developed a two-stage threading strategy: (a) threading using dynamic programming without considering the pairwise energy and (b) fold recognition considering all the energy terms, including the pairwise energy calculated from the dynamic programming threading alignments. (iii) We have developed a combined z-score scheme for fold recognition, which takes into consideration the z-scores of each energy term. (iv) Based on the z-scores, we have developed a confidence index, which measures the reliability of a prediction and a possible structure-function relationship based on a statistical analysis of a large data set consisting of threadings of 600 query proteins against the entire FSSP templates. Tests on several benchmark sets indicate that the evolutionary information and other new features of PROSPECT II greatly improve the alignment accuracy. We also demonstrate that the performance of PROSPECT II on fold recognition is significantly better than any other method available at all levels of similarity. Improvement in the sensitivity of the fold recognition, especially at the superfamily and fold levels, makes PROSPECT II a reliable and fully automated protein structure and function prediction program for genome-scale applications.","PeriodicalId":20902,"journal":{"name":"Protein engineering","volume":"16 9","pages":"641-50"},"PeriodicalIF":0.0,"publicationDate":"2003-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzg081","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24028194","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 103

Prediction of protein secondary structure with a reliability score estimated by local sequence clustering. 用局部序列聚类估计可靠性评分的蛋白质二级结构预测。

Protein engineering Pub Date : 2003-09-01 DOI: 10.1093/protein/gzg089

Fan Jiang

{"title":"Prediction of protein secondary structure with a reliability score estimated by local sequence clustering.","authors":"Fan Jiang","doi":"10.1093/protein/gzg089","DOIUrl":"https://doi.org/10.1093/protein/gzg089","url":null,"abstract":"Most algorithms for protein secondary structure prediction are based on machine learning techniques, e.g. neural networks. Good architectures and learning methods have improved the performance continuously. The introduction of profile methods, e.g. PSI-BLAST, has been a major breakthrough in increasing the prediction accuracy to close to 80%. In this paper, a brute-force algorithm is proposed and the reliability of each prediction is estimated by a z-score based on local sequence clustering. This algorithm is intended to perform well for those secondary structures in a protein whose formation is mainly dominated by the neighboring sequences and short-range interactions. A reliability z-score has been defined to estimate the goodness of a putative cluster found for a query sequence in a database. The database for prediction was constructed by experimentally determined, non-redundant protein structures with <25% sequence homology, a list maintained by PDBSELECT. Our test results have shown that this new algorithm, belonging to what is known as nearest neighbor methods, performed very well within the expectation of previous methods and that the reliability z-score as defined was correlated with the reliability of prediction. This led to the possibility of making very accurate predictions for a few selected residues in a protein with an accuracy measure of Q3 > 80%. The further development of this algorithm, and a nucleation mechanism for protein folding are suggested.","PeriodicalId":20902,"journal":{"name":"Protein engineering","volume":"16 9","pages":"651-7"},"PeriodicalIF":0.0,"publicationDate":"2003-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzg089","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24028195","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 15

Involvement of surface cysteines in activity and multimer formation of thimet oligopeptidase. 表面半胱氨酸参与硫寡肽酶的活性和多聚体形成。

Protein engineering Pub Date : 2003-08-01 DOI: 10.1093/protein/gzg073

J A Sigman, M L Sharky, S T Walsh, A Pabon, M J Glucksman, A J Wolfson

{"title":"Involvement of surface cysteines in activity and multimer formation of thimet oligopeptidase.","authors":"J A Sigman, M L Sharky, S T Walsh, A Pabon, M J Glucksman, A J Wolfson","doi":"10.1093/protein/gzg073","DOIUrl":"https://doi.org/10.1093/protein/gzg073","url":null,"abstract":"Thimet oligopeptidase is a metalloenzyme involved in regulating neuropeptide processing. Three cysteine residues (246, 248, 253) are known to be involved in thiol activation of the enzyme. In contrast to the wild-type enzyme, the triple mutant (C246S/C248S/C253S) displays increased activity in the absence of dithiothreitol. Dimers, purportedly formed through cysteines 246, 248 and 253, have been thought to be inactive. However, analysis of the triple mutant by native gel electrophoresis reveals the existence of dimers and multimers, implying that oligomer formation is mediated by other cysteines, probably on the surface, and that some of these forms are enzymatically active. Isolation and characterization of iodoacetate-modified monomers and dimers of the triple mutant revealed that, indeed, certain dimeric forms of the enzyme are still fully active, whereas others show reduced activity. Cysteine residues potentially involved in dimerization were identified by modeling of thimet oliogopeptidase to its homolog, neurolysin. Five mutants were constructed; all contained the triple mutation C246S/C248S/C253S and additional substitutions. Substitutions at C46 or C682 and C687 prevented multimer formation and inhibited dimer formation. The C46S mutant had enzymatic activity comparable to the parent triple mutant, whereas that of C682S/C687S was reduced. Thus, the location of intermolecular disulfide bonds, rather than their existence per se, is relevant to activity. Dimerization close to the N-terminus is detrimental to activity, whereas dimerization near the C-terminus has little effect. Altering disulfide bond formation is a potential regulatory factor in the cell owing to the varying oxidation states in subcellular compartments and the different compartmental locations and functions of the enzyme.","PeriodicalId":20902,"journal":{"name":"Protein engineering","volume":"16 8","pages":"623-8"},"PeriodicalIF":0.0,"publicationDate":"2003-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzg073","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22571231","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 13

Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface. 亚基界面van der Waals体积减小导致3-异丙基苹果酸脱氢酶不可逆失活，提高了热稳定性。

Protein engineering Pub Date : 2003-08-01 DOI: 10.1093/protein/gzg071

Takatoshi Ohkuri, Akihiko Yamagishi

{"title":"Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface.","authors":"Takatoshi Ohkuri, Akihiko Yamagishi","doi":"10.1093/protein/gzg071","DOIUrl":"https://doi.org/10.1093/protein/gzg071","url":null,"abstract":"We have investigated factors affecting stability at the subunit-subunit interface of the dimeric enzyme 3-isopropylmalate dehydrogenase (IPMDH) from Bacillus subtilis. Site-directed mutagenesis was used to replace methionine 256, a key residue in the subunit interaction, with other amino acids. Thermal stability against irreversible inactivation of the mutated enzymes was examined by analyzing the residual activity after heat treatment. The mutations M256V and M256A increased thermostability by 2.0 and 6.0 degrees C, respectively, whereas the mutations M256L and M256I had no effect. Thermostability of the M256F mutated enzyme was 4.0 degrees C lower than that of the wild-type enzyme. To our surprise, increasing the hydrophobicity of residue 256 within the hydrophobic core of the enzyme resulted in a lower thermal stability. The mutated enzymes showed an inverse correlation between thermostability and the volume of the side chain at position 256. Based on the X-ray crystallographic structure of Escherichia coli IPMDH, the environment around M256 in the B.subtilis homolog is predicted to be sterically crowded. These results suggest that Met256 prevents favorable packing. Introduction of a smaller amino acid at position 256 improves the packing and stabilizes the dimeric structure of IPMDH. The van der Waals volume of the amino acid residue at the hydrophobic subunit interface is an important factor for maintaining the stability of the subunit-subunit interface and is not always optimized in the mesophilic IPMDH enzyme.","PeriodicalId":20902,"journal":{"name":"Protein engineering","volume":"16 8","pages":"615-21"},"PeriodicalIF":0.0,"publicationDate":"2003-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzg071","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22571230","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

Optimizing the search algorithm for protein engineering by directed evolution. 定向进化优化蛋白质工程搜索算法。

Protein engineering Pub Date : 2003-08-01 DOI: 10.1093/protein/gzg077

Richard Fox, Ajoy Roy, Sridhar Govindarajan, Jeremy Minshull, Claes Gustafsson, Jennifer T Jones, Robin Emig

{"title":"Optimizing the search algorithm for protein engineering by directed evolution.","authors":"Richard Fox, Ajoy Roy, Sridhar Govindarajan, Jeremy Minshull, Claes Gustafsson, Jennifer T Jones, Robin Emig","doi":"10.1093/protein/gzg077","DOIUrl":"https://doi.org/10.1093/protein/gzg077","url":null,"abstract":"An in silico protein model based on the Kauffman NK-landscape, where N is the number of variable positions in a protein and K is the degree of coupling between variable positions, was used to compare alternative search strategies for directed evolution. A simple genetic algorithm (GA) was used to model the performance of a standard DNA shuffling protocol. The search effectiveness of the GA was compared to that of a statistical approach called the protein sequence activity relationship (ProSAR) algorithm, which consists of two steps: model building and library design. A number of parameters were investigated and found to be important for the comparison, including the value of K, the screening size, the system noise and the number of replicates. The statistical model was found to accurately predict the measured activities for small values of the coupling between amino acids, K <or= 1. The ProSAR strategy was found to perform well for small to moderate values of coupling, 0 <or= K <or= 3, and was found to be robust to noise. Some implications for protein engineering are discussed.","PeriodicalId":20902,"journal":{"name":"Protein engineering","volume":"16 8","pages":"589-97"},"PeriodicalIF":0.0,"publicationDate":"2003-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzg077","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22571227","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 65

Molecular dynamics simulations of the unfolding of beta(2)-microglobulin and its variants. β(2)-微球蛋白及其变体展开的分子动力学模拟。

Protein engineering Pub Date : 2003-08-01 DOI: 10.1093/protein/gzg079

Buyong Ma, Ruth Nussinov

{"title":"Molecular dynamics simulations of the unfolding of beta(2)-microglobulin and its variants.","authors":"Buyong Ma, Ruth Nussinov","doi":"10.1093/protein/gzg079","DOIUrl":"https://doi.org/10.1093/protein/gzg079","url":null,"abstract":"In this study, we examined the unfolding processes of native beta(2)-microglobulin and two related variants, one with an N-terminal hexapeptide deletion DeltaN6 and another with Lys57-Asp58 cleavage, by high-temperature molecular dynamics simulations. Three simulation models were used, molecular dynamics (MD) simulations with explicit water solvation, MD simulations with the CHARMM EEF1 force field and Langevin dynamics with the CHARMM EEF1 force field. Our simulations reproduce many of the experimentally observed structural changes. The most striking agreement is in the beta-strands to alpha-helix transition. In our simulations, strands beta(3), beta(4) and beta(5) consistently change to alpha-helix, whereas beta(8) changes to an alpha-helix only briefly. Through comparisons of the conformational behavior of the native, the DeltaN6 and the Lys-cut beta(2)-m, using the three simulation methods, we identified the consensus conformational changes that differentiate between the native beta(2)-m and its two variants. We found that the main effect of the removal of the N-terminal hexapeptide is to increase the separation between strands beta(2) and beta(6) and to facilitate the beta to alpha transition. On the other hand, the lysine cleavage only increases the flexibility of strand beta(5) and does not affect the interactions between strands beta(2) and beta(6). These conformational changes may relate to polymerization tendencies of these variants.","PeriodicalId":20902,"journal":{"name":"Protein engineering","volume":"16 8","pages":"561-75"},"PeriodicalIF":0.0,"publicationDate":"2003-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzg079","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22570768","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 29

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase. 保守的Pro196、Pro197和His199在胸苷酸合酶机制中的结构作用。

Protein engineering Pub Date : 2003-08-01 DOI: 10.1093/protein/gzg076

Dolores Gonzalez-Pacanowska, Luis M Ruiz-Perez, María Angeles Carreras-Gómez, Maria Paola Costi, Robert M Stroud, Janet Finer-Moore, Daniel V Santi

{"title":"The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase.","authors":"Dolores Gonzalez-Pacanowska, Luis M Ruiz-Perez, María Angeles Carreras-Gómez, Maria Paola Costi, Robert M Stroud, Janet Finer-Moore, Daniel V Santi","doi":"10.1093/protein/gzg076","DOIUrl":"https://doi.org/10.1093/protein/gzg076","url":null,"abstract":"We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198. Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity. To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199. All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity. For all His199 mutations, k(cat)/K(m) for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis. Pro196 can be substituted with small hydrophilic residues with little loss in k(cat), but 15- to 23-fold increases in K(m)(dUMP). Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower k(cat)/K(m)(dUMP) than wild-type TS. Several Pro196 and Pro197 variants were temperature sensitive. The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro-Pro sequence are largely maintained when either member of the pair is mutated.","PeriodicalId":20902,"journal":{"name":"Protein engineering","volume":"16 8","pages":"607-14"},"PeriodicalIF":0.0,"publicationDate":"2003-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzg076","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22571229","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 6

Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution. 通过定向进化提高南极念珠菌脂肪酶B对不可逆热失活的耐受性。

Protein engineering Pub Date : 2003-08-01 DOI: 10.1093/protein/gzg074

Ningyan Zhang, Wen-Chen Suen, William Windsor, Li Xiao, Vincent Madison, Aleksey Zaks

{"title":"Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution.","authors":"Ningyan Zhang, Wen-Chen Suen, William Windsor, Li Xiao, Vincent Madison, Aleksey Zaks","doi":"10.1093/protein/gzg074","DOIUrl":"https://doi.org/10.1093/protein/gzg074","url":null,"abstract":"To expand the functionality of lipase B from Candida antarctica (CALB) we have used directed evolution to create CALB mutants with improved resistance towards irreversible thermal inactivation. Two mutants, 23G5 and 195F1, were generated with over a 20-fold increase in half-life at 70 degrees C compared with the wild-type CALB (WT-CALB). The increase in half-life was attributed to a lower propensity of the mutants to aggregate in the unfolded state and to an improved refolding. The first generation mutant, 23G5, obtained by error-prone PCR, had two amino acid mutations, V210I and A281E. The second generation mutant, 195F1, derived from 23G5 by error-prone PCR, had one additional mutation, V221D. Amino acid substitutions at positions 221 and 281 were determined to be critical for lipase stability, while the residue at position 210 had only a marginal effect. The catalytic efficiency of the mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyl octanoate was also found to be superior to that of WT-CALB.","PeriodicalId":20902,"journal":{"name":"Protein engineering","volume":"16 8","pages":"599-605"},"PeriodicalIF":0.0,"publicationDate":"2003-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzg074","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22571228","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 183

Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome p450 monooxygenase. 昆虫细胞色素p450单加氧酶CYP6B1催化位点底物和抑制剂的分子对接。

Protein engineering Pub Date : 2003-08-01 DOI: 10.1093/protein/gzg075

Jerome Baudry, Weimin Li, Liping Pan, May R Berenbaum, Mary A Schuler

{"title":"Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome p450 monooxygenase.","authors":"Jerome Baudry, Weimin Li, Liping Pan, May R Berenbaum, Mary A Schuler","doi":"10.1093/protein/gzg075","DOIUrl":"https://doi.org/10.1093/protein/gzg075","url":null,"abstract":"Furanocoumarins represent plant toxins that are used in the treatment of a variety of skin diseases and are metabolized by cytochrome p450 monooxygenases (p450s) existing in insects such as Papilio polyxenes (the black swallowtail). To elucidate the active site in the CYP6B1 protein that is the principal p450 existing in this species, we have constructed a homology model of it based on sequence and structure alignments with the bacterial CYP102 protein whose crystal structure has been defined and with the insect CYP6B4 protein that also metabolizes furanocoumarins. In the derived CYP6B1 model, Phe116 and His117 in SRS1, Phe371 in SRS5 and Phe484 in SRS6 contribute to the formation of a resonant network that stabilizes the p450's catalytic site and allows for interactions with its furanocoumarin substrates. The first two of these residues are absolutely conserved in all members of the insect CYP6B subfamily and the last two are variable in different members of the CYP6B subfamily. A combination of theoretical and experimental docking analyses of two substrates (xanthotoxin and bergapten) and two inhibitors (coumarin and pilocarpine) of this p450 provide significant information on the positioning of furanocoumarins within this catalytic pocket. Molecular replacement models based on the results of variations at two of these critical amino acids provide support for our furanocoumarin-docked model and begin to rationalize the altered substrate reactivities observed in experimental analyses.","PeriodicalId":20902,"journal":{"name":"Protein engineering","volume":"16 8","pages":"577-87"},"PeriodicalIF":0.0,"publicationDate":"2003-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzg075","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22570769","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 70