X-RAY DIFFRACTION STUDIES OF THE HG(II)-SPECIFIC TRANSCRIPTIONAL REGULATOR MERR

HG(II) 特异性转录调控因子 MERR 的 X 射线衍射研究

• 批准号: 8363692
• 负责人: STEPHEN TOMANCIEK
• 金额: $ 0.61万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363692
• 关键词: Bacteria; Binding; Computer Simulation; DNA; Data; Data Quality; Ecosystem; Elements; Family; Funding; Genes; Genetic Transcription; Goals; Grant; Homology Modeling; Mercury; Metals; Modeling; Molecular; National Center for Research Resources; Neutrons; Operon; Principal Investigator; Research; Research Infrastructure; Resistance; Resolution; Resources; Roentgen Rays; Source; Structural Models; Structure; Transcriptional Regulation; United States National Institutes of Health; X ray diffraction analysis; X-Ray Diffraction; bacterial resistance; beamline; cost; high end computer; interest; member; molecular dynamics; multidisciplinary; protein structure; research study; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of our research is to apply structural biology and high performance computer simulation to investigate molecular mechanisms of bacterial resistance to mercury. Specifically we are interested in solving the first ever X-ray crystal structure of the metal-responsive transcriptional regulator MerR. MerR is the archetype of the MerR family of metalloregulators that controls the transcription of a set of genes (the mer operon) providing Hg resistance in many genera of bacteria isolated from Hg-exposed ecosystems. The mer operon encodes specific genes that facilitate transport of Hg species cleavage of organomercurials and reduction of ionic Hg(II) to volatile elemental Hg(0). The 144-residue MerR binds to its operator DNA and functions as a homodimer. It represses transcription of the mer operon in the absence of Hg(II) and activates transcription upon Hg(II) binding. We wish to obtain a high resolution model to fully characterize Hg(II) binding to MerR. This structural model of MerR will support ongoing neutron scattering and Molecular Dynamics (MD) experiments aimed at studying MerR dynamics and conformational changes upon Hg(II) binding that are essential for understanding its unique mechanism of transcriptional regulation. It is well-known that these multidisciplinary structural studies require maximal resolution X-ray structures of the protein element. As a result a high flux beamline is required to collect high quality data for this proposed research. One day of beamtime (3 shifts) is requested to complete the proposed diffraction studies. We plan to screen crystals of MerR for X-ray diffraction quality and to collect complete redundant data at the highest resolution possible. Structure determination will then be completed by molecular replacement using either a known structure of a paralogous member of the MerR family or by using our current homology model of MerR.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们的研究目标是应用结构生物学和高性能计算机模拟来研究细菌对汞抗性的分子机制。具体来说，我们有兴趣解决有史以来第一个X射线晶体结构的金属响应转录调节MerR。MerR是MerR家族金属调节因子的原型，其控制一组基因（mer操纵子）的转录，在从汞暴露生态系统分离的许多属的细菌中提供汞抗性。mer操纵子编码促进汞物种运输、有机汞化合物裂解和离子汞（II）还原为挥发性元素汞（0）的特定基因。144个残基的MerR与其操纵基因DNA结合，并作为同源二聚体发挥作用。在没有Hg（II）的情况下，它抑制mer操纵子的转录，并在Hg（II）结合时激活转录。我们希望获得一个高分辨率的模型，以充分表征汞（II）结合MerR。这种结构模型的MerR将支持正在进行的中子散射和分子动力学（MD）的实验，旨在研究MerR的动力学和构象变化后，汞（II）的结合，是必不可少的理解其独特的转录调控机制。众所周知，这些多学科的结构研究需要最大分辨率的蛋白质元素的X射线结构。因此，高通量光束线是需要收集高质量的数据，这项拟议的研究。需要一天的光束时间（3个班次）来完成拟议的衍射研究。我们计划筛选MerR晶体的X射线衍射质量，并以最高分辨率收集完整的冗余数据。 然后，使用MerR家族旁系同源成员的已知结构或使用我们目前的MerR同源模型通过分子置换完成结构测定。