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Chinese Journal of Structural Chemistry 2019年第3期38卷 422-428页

作者： ZOU Xiao-Ming JIANG Mei-Qin ZHAO Yan-He SUN Li-Fang YANG Wen-Di YE Xian-Ren WU Yun-Kun State Key Laboratory of Structural Chemistry Fujian Institute of Research on the Structure of Matter Chinese Academy of Sciences Department of Cell Biology and Biophysics University of Texas Southwestern Medical Center Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation College of Life Science Fujian Normal University Fujian Cancer Hospital & Fujian Medical University Cancer Hospital

The lincosamide class of antibacterials is widely used for the treatment of a broad spectrum of infections, and one prevalent route of resistance to lincosamides in pathogenic gram-positive cocco is antibiotic modification. Enzymes encoded by lin genes, belonging to nucleotidyltransferase superfamily, catalyze adenylylation to inactivate lincosamides. LinA can adenylylate lincosamides at either 3?-or 4?-OH of the methylthiolincosamide sugar. The crystal structure of LinA/lincomycin has confirmed its active site. However, the residue interacting with nucleotidyl donors remains elusive. Here, we modeled the complex structure of LinA/lincomycin/Mg^(2+)/AMPCPP to reveal a putative pocket for nucleotidyl donors and suggested the residue R45 in this pocket involved in the recognition of donor substrates NTP and catalysis. ITC and enzyme activity assays show that the mutation of residue R45 impairs LinA nucleotidyltransferase activity in vitro. This work provides insights into the molecular mechanism of the nucleotide binding and transferring activity of antibiotic NTases.

关键词： LinA nucleotidyltransferase site-directed mutagenesis enzymatic assay ITC

Molecular dynamics simulation of site-directed mutagenesis of HIV-1 Tat trans-activator

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Chinese Science Bulletin 1999年第8期44卷 708-711页

作者： Yan Cui Lunjiang Ling Runsheng Chen Longchuan Bai Jiangang Yuan Boqin Qiang Chinese Acad Sci Inst Biophys Beijing 100101 Peoples R China Chinese Acad Med Sci Inst Basic Med Sci Beijing 100005 Peoples R China

The Cys-rich domain, core region and basic domain are highly conserved and very important to the trans-activation activity of HIV-1 Tat trans-activator. The three-dimensional structures of 6 mutants of HIV-1 Tat protein were constructed with the methods of molecular dynamics simulation. The variations of the structures of the mutants have been analyzed and the factors that led to abolishment of trans-activation activity have been discussed.

关键词： human immunodeficiency virus type 1 ( HIV-1) molecular dynamics simulation site-directed mutagenesis Tat protein.

Monitoring the autoproteolysis of hiv-1 protease by site-directed spin-labeling and electron paramagnetic resonance spectroscopy

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Journal of Biophysical Chemistry 2011年第2期2卷 137-146页

作者： Jamie L. Kear Luis Galiano Angelo M. Veloro Laura S. Busenlehner Gail E. Fanucci Department of Chemistry University of Florida Gainesville Florida USA

site-directed spin-labeling with continuous wave electron paramagnetic resonance spectroscopy was used to monitor autoproteolysis of HIV-1 protease, an enzyme essential for viral maturation. Two protein constructs were examined, namely subtype F and the circulating recombinant form CRF01_A/E. As the protease undergoes self-cleavage, protein unfolds and small peptide fragments containing the spin label are generated, which collectively give rise to a sharp spectral component that is easily discernable in the high-field resonance line in the EPR spectrum. By monitoring the intensity of this spectral component over time, the autoproteolytic stability of each construct was characterized under various conditions. Data were collected for samples stored at 4 °C, 25 °C, and 37 °C, and on a subtype F HIV-1 protease sample stored at 25 °C and containing the FDA-approved protease inhibitor Tipranavir. As expected, the rate of autoproteolysis decreased as the storage temperature was lowered. Minimal autoproteolysis was seen for the sample that contained Tipranavir, providing direction for future spectroscopic studies of active protease samples. When compared to standard methods of monitoring protein degradation such as gel electrophoresis or chromatographic analyses, spin-labeling with CW EPR offers a facile, real-time, non-consuming way to monitor autoproteolysis or protein degradation. Additionally, mass spectrometry studies revealed that the N-termini of both constructs are sensitive to degradation and that the sites of specific autoproteolysis vary.

关键词： HIV-1 Protease Autoproteolysis Self-Proteolytic Activity site-directed Spin-Labeling Electron Paramagnetic Resonance (EPR) Spectroscopy

Construction and screening of a multi-point site- specific mutant library of subtilisin E with a set of oligonucleotides

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Science China(Life Sciences) 1997年第4期40卷 337-344页

作者：肖谷田谢毅张婕孙筱清吴小舟陈小央王启松 State Key Laboratory of Genetic Engineering Institute of Genetics Fudan University Shanghai 200433 China

A mutant library of subtilisin E containing random combinations of various mutagenized sites wasconstructed by one-round mutagenesis with 15 mutagenic oligonucleotides. Mutants were screened through dot blot hybridization and DNA sequencing. A single-point mutant (Met 222Ala) and a three-point (Asn 76Asp/Asnl09Ser/ I le 205/Cys) mutant gene from the library were expressed. The mutant proteins exhibited conspicuously improved resistance to oxidation and heat treatment, as reported before. The results show that the library is reliable and very useful for protease subtilisin E engineering.

关键词： site-directed mutagenesis multi-point site-specific mutagenesis protein engineering subtilisin E.

Construction of novel tumor necrosis factor-alpha mutants with reduced toxicity and higher cytotoxicity on human tumor cells

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Science China(Life Sciences) 2003年第1期46卷 1-9页

作者：刘惠卢芳陈建军任红玉陈常庆 Shanghai Research Center of Biotechnology Chinese Academy of Sciences

Two tumor necrosis factor-a mutants MT1 (32Trp157Phe) and MT2 (2Lys30Ser- 32Trp157Phe) were constructed by site-directed mutagenesis. These mutants were soluble and over-expressed in E. coli. The purity of purified mutants was above 95% by serial chromatography. The results of Western blot indicated that these mutants could be cross-reactive with monoclonal antibody against native hTNF-a. Compared to parent hTNF-a, the cytotoxicity of these mutants on murine fibrosarcoma L929 cell lines reduced 4—5 orders of magnitude but was equivalent to that of native hTNF-a on human tumor cell lines. The LD50 of mutant MT1 was reduced to 0.34% of wild type and the dose of MT2 that resulted in 30% death of mice reduced to less than 1/700 that of parent hTNF-a.

关键词： human tumor necrosis factor, site-directed mutation, cytotoxicity, LD50, Western blot.

A urokinase mutant with high fibrin-selectivity

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Chinese Science Bulletin 1997年第11期42卷 942-947页

作者： Peng, GH Ma, Z Yu, RR Xue, YM Zhu, DX NANJING UNIV DEPT BIOCHEMSTATE KEY LAB PHARMACEUT BIOTECHNOLNANJING 210093PEOPLES R CHINA

BECAUSE of lacking fibrin selectivity two chain urokinase-type plasminogen activator(tcu-PA),which has been used as the clinical thrombolytic agent,may lead to bleeding by a remarkabledepletion of fibrinogen in plasma when injected in large doses,thus its clinical application *** chain urokinase-type plasminogen activator(scu-PA)is the precursor of tcu-

关键词： scu-PA tcu-PA site-directed mutagenesis fibrin selectivity.

Important amino acid in the hemagglutinin glycoprotein of measles virus(MV)that governs hemadsorption

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Chinese Science Bulletin 1999年第7期44卷 612-617页

作者： Michiko Watanabe Shigeharu Ueda Institute of Microbial Diseases Osaka University Osaka 565Institute of Microbial Diseases Osaka University Osaka 565 JapanJapan

Two strains of measles virus IMA and SMD that were isolated using B95a cell line show hemadsorption negative. After adaptation to Vero cells, these strains gain the ability to agglutinate AGM-RBC and show hemadsorption positive. Sequence analysis of these two kinds of virus reveals that the two strains all have a Ser (HAD negative) to Gly (HAD positive) mutation at position 546 in the H protein. site-directed mutagenesis and expression in COS cells were used to confirm that the mutation Ser→Gly is responsible for hemadsorption alteration. Then the monoclonal antibody against CD46 was used to identify that this mutation also governs the binding function of MV H protein to CD46 receptor. The data provide a new important amino acid of MV H protein that governs the hemadsorption and binding to CD46 receptor.

关键词： H protein CD46 receptor hemadsorption site-directed mutagenesis.

Preparation and characterization of some surface negatively charged residue mutants of cytochrome b_5

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Chinese Science Bulletin 2001年第7期46卷 555-559页

作者： WANG Yunhua, WANG Wenhu, LU Junxia, REN Yi, GU Shaohua, XIE Yi & HUANG Zhongxian1. Chemical Biology Lab, Department of Chemistry, Fudan University, Shanghai 200433, China 2. Genetic Institute, School of Life, Fudan University, Shanghai 200433, China Chemical Biology Lab Department of Chemistry Fudan University Shanghai China Genetic Institute School of Life Fudan University Shanghai China

site-directed mutagenesis was used to obtain seven variants of tryptic fragment of bovine liver cytochrome bs (cyt bs), in which the negatively charged residues around the heme exposed edge of cyt bs were replaced by hydropho-bic amino acid alanine. Double-site mutants, triple-site mutants and even quadruple-site mutants were obtained. DNA sequencing and molecular weight measurements of the mutant proteins both confirmed that these site-directed muta-genesises were successfully performed. Spectroelectrochem-istry of these mutant proteins revealed that the apparent redox potentials of these mutant proteins caused a positive shift of 2-10 mV. The global structure of these mutant proteins did not show much difference from that of the wild type cyt bs, providing a solid base for the further study on the roles of the proteins’ surface charges.

关键词： cytochrome bs site-directed mutagenesis negative charge on the protein surface redox potential.

Effect of different immunosuppressive drugs on calcineurin and its mutants

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Science China(Life Sciences) 2000年第1期43卷 68-74页

作者：阎力君于翠娟张丽芳魏群 Department of Biochemistry and Molecular Biology Beijing Normal University Beijing 100875 China

Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis. Their phosphatase activity and the corresponding solution conformation were examined. Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immuno-suppressive drugs with CN. The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN. Furthermore, circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein. Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN, and had no effects on the phosphatase activity of mutants in Loop7 region, which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN. Examination of the activities of these mutants resulted in the presence of immunosuppressive component from traditional Chinese drugs. The component of Chinese drug, ZIP1, could directly inhibit both CN and CN mutants without drug binding protein. These results suggest that the Loop7 region is an important structural area involved in the inhibition by CyP-CsA. It is valuable to further study the inhibition by ZIP1.

关键词： calcineurin site-directed mutagenesis immunosuppressive drugs circular dichroism spectrum.

A Single Mutation in the Hepta-Peptide Active site of <i>Aspergillus niger</i>PhyA Phytase Leads to Myriad Biochemical Changes

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Advances in Microbiology 2012年第3期2卷 388-394页

作者： Abul H. J. Ullah Kandan Sethumadhavan Stephanie Boone Edward J. Mullaney Southern Regional Research Center Agricultural Research Service United States Department of Agriculture New Orleans USA

The active site motif of proteins belonging to "Histidine Acid Phosphatase" (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger PhyA phytase. However, another phytase, PhyB, from the same microorganism has a higher turnover number and it shows E in this position. We mutated A69 residue to E in the fungal PhyA phytase. The mutant phytase shows a myriad of new kinetic properties. The pH profile shifted 0.5 pH unit in both 5.0 and 2.5 bi-hump peaks. The optimum temperature shifted down from 58℃ to 55℃. However, the greatest difference was observed in the mutant protein's reaction to GuCl at a concentration of 0.1 to 0.2 M. The activity of the mutant phytase jumped 100% while the wild type protein showed no activity enhancement in the same concentration range of GuCl. The kinetics performed at higher concentration of GuCl also contrasted the difference between the wild type and mutant phytase. While Km was least affected, the Vmax increased for the mutant and decreased for the wild type. The sensitivity towards myo-inositol hexasulfate, a potent inhibitor, was decreased by the mutation. All in all, A69E mutation has affected a multitude of enzymatic properties of the protein even though the residue was thought to be non-critical for phytase's catalytic function notwithstanding its location in the conserved hepta-peptide region of the biocatalyst.

关键词： Phytase Histidine Acid Phosphatase Aspergillus niger site-directed Mutagenesis