PROBING NITROGENASE FE PROTEIN NUCLEOTIDE DEPENDENT CONFORMATIONAL CHANGES USING

使用 探测固氮酶 FE 蛋白质核苷酸依赖的构象变化

• 批准号: 7954363
• 负责人: JOHN W PETERS
• 金额: $ 0.02万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954363
• 关键词: Binding; Complement; Computer Retrieval of Information on Scientific Projects Database; Data Collection; Funding; Grant; Hydrolysis; Institution; Investigation; MgADP; MgATP; Molecular Conformation; Nucleotides; Proteins; Research; Research Personnel; Resources; Role; Solutions; Source; Structure; Time; United States National Institutes of Health; Variant; X ray diffraction analysis; X-Ray Diffraction; aqueous; base; interest; nitrogenase reductase; nucleoside triphosphate; simulation; structural biology; synchrotron radiation

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. X-ray diffraction studies conducted by our group and others have revealed that the nitrogenase Fe protein can exist in a number of conformations; however, not all conformational states of the Fe protein of interest, in terms of defining the role of MgATP binding and hydrolysis, have been characterized. The key missing structure in defining nucleotide dependent conformation change in the Fe proteins is the Fe protein with bound MgATP. Determining the structure of a protein that undergoes nucleotide dependent conformational change in the presence of bound nucleoside triphosphates is challenging, partially attributable to the fact that nucleoside triphosphates in aqueous solutions are rapidly hydrolyzed under most circumstances over the course of the time it takes to obtain protein crystals. We therefore intend to conduct solution studies using SAXS to complement our X-ray diffraction studies. Simulations based on known structures reveal strikingly different scattering curves in the 1/q range of 0.15 and 0.25 for the Fe protein with MgADP bound and the Fe protein variant presumed to represent a mimic of the native MgATP bound state. This provides the basis for examining whether the conformation of the native Fe protein with MgATP bound resembles in conformation the Fe protein with MgADP bound or the Fe protein variant assigned as a putative MgATP bound mimic. Preliminary investigations at SSRL on Beam Line 4-2 have provided the proof-of-concept and demonstrated that the proposed studies are feasible. We are requesting approval for beamtime to further optimize our data collection strategies, examine nucleotide bound states directly.

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。 我们小组和其他人进行的X射线衍射研究表明，氮酶Fe蛋白可以在许多构象中存在。但是，在定义MGATP结合和水解的作用方面，并非所有感兴趣的Fe蛋白的构象状态都被表征了。 Fe蛋白中定义核苷酸依赖性构象变化的关键缺失结构是具有结合MGATP的Fe蛋白。在结合核苷三磷酸盐的存在下确定经历核苷酸依赖构象变化的蛋白质的结构是具有挑战性的，部分归因于以下事实：在大多数情况下，在大多数情况下，在获得蛋白质晶体的过程中，水溶液中的核苷三磷酸盐在水溶液中迅速水解。因此，我们打算使用SAX进行解决方案研究，以补充我们的X射线衍射研究。基于已知结构的仿真显示，具有MGADP结合的Fe蛋白的1/Q范围为0.15和0.25的散射曲线，而Fe蛋白变体被认为代表了天然MGATP结合态的模仿。这为检查与MGATP结合的天然Fe蛋白的构象相似于与MGADP结合的Fe蛋白的构象是否相似，还是将Fe蛋白变体或分配为推定的MGATP结合模拟的Fe蛋白变体。 SSRL在光束线4-2上的初步研究提供了概念验证，并证明了拟议的研究是可行的。我们要求批准Beamtime，以进一步优化我们的数据收集策略，直接检查核苷酸结合状态。