XAS STUDIES OF NOVEL ARSENIC BINDING SITES IN AS (III)-RESPONSIVE TRANSCRIPTIONA

AS (III) 响应转录中新型砷结合位点的 XAS 研究

• 批准号: 7722025
• 负责人: BARRY P. ROSEN
• 金额: $ 0.23万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722025
• 关键词: Arsenic; Bacteria; Binding; Binding Sites; C-terminal; Computer Retrieval of Information on Scientific Projects Database; Corynebacterium glutamicum; Cysteine; DNA Binding Domain; Drug Metabolic Detoxication; Funding; Genes; Glutamic Acid; Goals; Gold; Grant; Hydroxyl Radical; Industry; Institution; Ligands; Location; Methyltransferase; Mining; Molecular Chaperones; Mutagenesis; N-terminal; Personal Satisfaction; Plasmids; Production; Proteins; Research; Research Personnel; Resources; Site; Source; Sulfuric Acids; Transcription Repressor/Corepressor; United States National Institutes of Health; mutant; novel

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this project is characterization of arsenic binding sites in proteins involved in arsenic detoxification. ArsRs are As(III)-responsive transcriptional repressors that regulate expression of arsenic detoxification genes. We have identified three homologous ArsRs from three different bacteria but apparently have evolved different arsenic binding sites in different locations in each protein. The first is the well-characterized ArsR from plasmid R773. The binding site in this repressor is an S3 site composed of three co-linear cysteine residues, Cys32, Cys34 and Cys37 in the DNA binding domain. The second is an ArsR from Acidothiobacillus ferrooxidans, a sulfuric acid-producing bacterium used in the gold mining industry. Preliminary EXAFS results suggest that the site in this ArsR is a mixed S/O/N site, suggesting an S2 site and a hydroxyl ligand. This repressor has a C-terminal vicinal cysteine pair residues that may form an As(III) binding site. The third ArsR is from In Corynebacterium glutamicum, which is used for the production of glutamic acid. This ArsR has an N-terminal vicinal cysteine pair and a third cysteine near the DNA binding domain that we hypothesize forms an S3 site for As(III). EXAFS of each of the three repressors with bound As(III) will differentiate between these possibilities. Mutant ArsRs lacking cysteine residues, singly and in combination, will be used to identify the specific ligands in each site. ArsD is a novel As(III) metallochaperone. It has three vicinal cysteine pairs and several other cysteines. Cys12,13 and 18 have been identified by mutagenesis as required for chaperone activity. ArsD mutants in these cysteine residues, singly and in combination, will be used to characterize the As(III) binding site. ArsM is a newly identified As(III)-SAM methyltransferase that methylates As(III) to a variety of species. Participation of conserved residues Cys30 and 31 in As(III) binding site will be investigated.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 该项目的总体目标是对参与砷排毒的蛋白质中砷结合位点的表征。 ARSR为（III） - 响应转录阻遏物，调节砷排毒基因的表达。我们已经鉴定出来自三种不同细菌的三个同源ARSR，但显然已经在每种蛋白质的不同位置进化了不同的砷结合位点。首先是质粒R773的特征良好的ARSR。该阻遏物中的结合位点是由三个共线性半胱氨酸残基Cys32，cys34和cys37组成的S3位点。第二个是来自酸硫硫杆菌铁氧化物的ARSR，这是一种用于黄金矿业行业的产生硫酸的细菌。初步的EXAFS结果表明，该ARSR中的位点是混合的S/O/N位点，表明S2位点和羟基配体。该阻遏物具有C末端囊肿的半胱氨酸对残基，该残基可能形成AS（III）结合位点。第三个ARSR来自谷氨酸核酸杆菌，用于生产谷氨酸。该ARSR具有N末端囊肿的半胱氨酸对，并且在DNA结合结构域附近的第三个半胱氨酸，我们假设该结合结构域形成了AS（III）的S3位点。具有绑定为（iii）的三个阻遏物中的每一个的EXAF将区分这些可能性。缺乏半胱氨酸残基的突变体ARSR将单独且结合使用，用于识别每个位点中的特定配体。 ARSD是（III）金属伴侣的小说。它具有三个替代性半胱氨酸对和其他几种半胱氨酸。 Cys12,13和18已通过诱变鉴定为伴侣活性所需的。这些半胱氨酸残基中的ARSD突变体（单独和组合）将用于表征AS（III）结合位点。 ARSM是一种新识别为（iii）-SAM甲基转移酶，将其甲基化为（III）到多种物种。将研究（III）结合位点的保守残基Cys30和31的参与。