PROBING NITROGENASE FE PROTEIN NUCLEOTIDE DEPENDENT CONFORMATIONAL CHANGES USING

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们的小组和其他人进行的X-射线衍射研究表明，固氮酶Fe蛋白可以存在于许多构象;然而，并不是所有的构象状态的Fe蛋白的利益，在定义的MgATP结合和水解的作用，已被其特征在于。定义Fe蛋白中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。确定在结合的核苷三磷酸存在下经历核苷酸依赖性构象变化的蛋白质的结构是具有挑战性的，部分归因于这样的事实，即在大多数情况下，核苷三磷酸在水溶液中在获得蛋白质晶体所需的时间内被快速水解。因此，我们打算使用SAXS进行溶液研究，以补充我们的X射线衍射研究。基于已知结构的模拟揭示了显着不同的散射曲线在1/q的范围为0.15和0.25的Fe蛋白与MgADP结合和Fe蛋白的变体假定代表一个模拟的天然MgATP结合状态。这为检查结合MgATP的天然Fe蛋白的构象是否与结合MgADP的Fe蛋白或指定为推定的MgATP结合模拟物的Fe蛋白变体的构象相似提供了基础。SSRL对光束线4-2的初步调查提供了概念验证，并证明了拟议的研究是可行的。我们正在申请批准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。

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.