CRYSTALLOGRAPHIC STUDIES OF METAL-DIRECTED SUPERPROTEIN ASSEMBLIES AND PROTON TR

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。 第一个项目的重点是通过金属配位化学产生离散的多蛋白质组件。尽管有广泛的研究，但控制蛋白质-蛋白质相互作用(PPI)的能力仍然是一个巨大的挑战，因为PPI是由分布在大表面上的许多弱的非共价键叠加而成的。我们在这个项目中的目标是利用金属-配体相互作用的强度、方向性和选择性来控制PPI，从而在不需要广泛结合表面的情况下实现特异性和亲和力。在利用四螺旋束蛋白细胞色素cb562作为构建块的初步实验中，我们已经证明了合理设计蛋白质表面的金属结合基序(MBMS)可以形成离散的多蛋白质结构，其低聚状态和几何结构完全由金属配位控制。这种方法不仅产生了复杂的生物组装，而且还产生了建立在蛋白质-蛋白质界面上的新的金属分配中心。到目前为止，我们已经收集了四个超蛋白组装体的结晶学数据集，使用的是主要为小分子结晶学设计的装置，这种装置产生的数据分辨率/质量有限。在这项提议中，我们的目标是使用可调谐的同步辐射来获得我们已经结晶的多达10个组件的高分辨率结构，彻底建立金属配位几何结构，并明确地确认其中所含金属的存在并确定它们的身份。第二个项目旨在绘制固氮酶钼铁蛋白(MoFeP)中潜在的质子转移途径，固氮酶是负责生物固氮的酶。MoFeP催化8电子/8质子还原氮气为氨。虽然对MoFeP中的电子转移有一定的了解，但质子转移途径还没有建立起来。使用一种已经成功的细胞色素C氧化酶和

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. The first project focuses on the generation of discrete multi-protein assemblies through metal coordination chemistry. Despite extensive research, the ability to control protein-protein interactions (PPIs) remains a great challenge, owing to the fact that PPIs are guided by the superposition of many weak, non-covalent bonds spread over large surfaces. Our goal in this project is to utilize the strength, directionality and selectivity of metal-ligand interactions to control PPIs, thereby achieving specificity and affinity without requiring extensive binding surfaces. In preliminary experiments utilizing the four-helix bundle protein cytochrome cb562 as a building block, we have demonstrated that rationally designed metal-binding-motifs (MBMs) on protein surfaces can nucleate the formation of discrete multi-protein structures, whose oligomeric states and geometries are controlled entirely by metal coordination. Not only does this approach yield complex bioassemblies, but also gives rise to novel metallocenters built within protein-protein interfaces. We have so far collected crystallographic data sets on four superprotein assemblies, using a setup designed primarily for small molecule crystallography, which has yielded limited data resolution/quality. In this proposal, we aim to employ tunable synchrotron radiation to obtain high-resolution structures of up to 10 assemblies that we have crystallized, thoroughly establish the metal coordination geometries, and unambiguously confirm the presence of the metals contained within and determine their identities. The second project aims to map potential proton-transfer pathways in the molybdenum-iron protein (MoFeP) of nitrogenase, the enzyme responsible for biological nitrogen fixation. MoFeP catalyzes the 8 electron/8 proton reduction of dinitrogen into ammonia. While electron transfer in MoFeP is somewhat well understood, proton transfer pathways have not been established. Using an approach that has been successful with cytochrome c oxidase and