PROBING NUCLEOTIDE DEPENDENT CONFORMATIONAL CHANGE IN THE NITROGENASE FE PROTEIN

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。 在固氮酶催化过程中，铁蛋白和钼-铁蛋白以一种方式结合和解离，导致两个分子的镁三磷酸腺苷的水解和至少一个电子转移到MoFe蛋白。这些依赖核苷酸的构象变化与其他一些生化信号转导机制的例子相似，因此，探讨核苷三磷酸结合和水解在固氮酶中的作用具有广泛的意义。有相当多的生化和光谱证据，包括小角X射线散射研究，表明铁蛋白在核苷酸置换过程中经历了构象变化(镁三磷酸腺苷或三磷酸核苷)。我们最近确定了铁蛋白的一个位点直接变体的结构，它在没有核苷酸的情况下具有许多生化和光谱特征，这些核苷酸类似于天然铁蛋白在结合的镁三磷酸腺苷存在下的存在。分离铁蛋白变体和上述铁蛋白结构的最显著的结构差异主要表现在铁蛋白亚基相互之间的刚体重新定位。每个铁蛋白亚基通过相对于先前表征的铁蛋白结构以60到80度的数量级彼此的刚体旋转来重定向。我们用晶体坐标计算表明，尽管变异体中缺少残基，但位点直接变异体的旋转半径约比天然铁蛋白的旋转半径大2.2°。这一结果显然与先前关于天然蛋白质与镁结合的SAXS结果相矛盾[Chen等人，(1994)J.Biol。化学。269,3290]。我们想要重新审视天然铁蛋白在核苷酸结合状态下的SAXS，以检验我们假设的铁蛋白构象变化模型的有效性。

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. During nitrogenase catalysis, the iron protein and molybdenum-iron protein associate and dissociate in a manner resulting in the hydrolysis of two molecules of MgATP and the transfer of at least one electron to the MoFe protein. These nucleotide dependent conformational changes are analogous to a number of other examples of biochemical signal transduction machinery and therefore probing the role of nucleoside triphosphate binding and hydrolysis in nitrogenase is of broad significance. There is considerable biochemical and spectroscopic evidence including small angle x-ray scattering studies that have implicate that the Fe protein undergoes conformational changes during nucleotide displacement (MgATP for MgADP or nucleoside triphosphate hydrolysis. We have recently determined the structure of a site-direct variant of the Fe protein that has a number of biochemical and spectroscopic features in the absence of nucleotide that resemble the native Fe protein in the presence of bound MgATP. The most significant structural differences that separate the Fe protein variant and the aforementioned structures of the Fe protein are largely manifested in a rigid body reorientation of the Fe protein subunits with respect to one another. Each of the Fe protein subunits is reoriented by a rigid-body rotation with respect to one another on the order of sixty to eighty degrees relative to the previously characterized Fe protein structures. Our calculation using the crystallographic coordinates indicates that the site-direct variant has a radius of gyration approximately 2.2 ¿ larger than that of the native Fe protein despite the missing residues in the variant. This result apparently contradicts the previous SAXS results on the native protein upon MgATP binding [Chen et al., (1994) J. Biol. Chem. 269, 3290]. We would like to revisit SAXS of the native Fe protein in nucleotide bound states to examine the validity of our hypothetical model for the conformational changes the Fe protein undergoes.