PROBING NITROGENASE FE PROTEIN NUCLEOTIDE DEPENDENT CONFORMATIONAL CHANGES USING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们和其他人进行的X射线衍射研究表明，固氮酶铁蛋白可以以多种构象存在；然而，就确定镁ATP结合和水解的作用而言，并不是所有感兴趣的铁蛋白的构象状态都得到了表征。确定铁蛋白中核苷酸依赖性构象变化的关键缺失结构是与镁三磷酸腺苷结合的铁蛋白。在结合的核苷三磷酸盐存在的情况下，确定发生核苷酸依赖构象变化的蛋白质的结构是具有挑战性的，部分原因是在大多数情况下，水溶液中的核苷三磷酸盐在获得蛋白质晶体的过程中会被迅速水解。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显示，带有镁ADP结合的铁蛋白和被认为代表天然镁ATP结合状态的铁蛋白变体在1/Q范围内的散射曲线在0.15和0.25范围内有显著不同。这为研究天然铁蛋白与镁ATP结合的铁蛋白的构象是否与与镁ADP结合的铁蛋白或被指定为可能的镁ATP结合模拟物的铁蛋白变体的构象相似提供了基础。在4-2号线上的SSRL的初步调查已经提供了概念验证，并证明拟议的研究是可行的。我们正在请求批准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。

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.