CRYSTALLOGRAPHIC STUDIES OF METAL-DIRECTED SUPERPROTEIN ASSEMBLIES AND PROTON TR

这个子项目是许多研究子项目中的一个 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。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 第一个项目的重点是通过金属配位化学生成离散的多蛋白质组装体。尽管进行了广泛的研究，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是由许多分布在大表面上的弱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，从而实现特异性和亲和力，而不需要广泛的结合表面。在初步实验中，利用四螺旋束蛋白质细胞色素cb 562作为积木，我们已经证明，合理设计的金属结合基序（MBMs）的蛋白质表面可以核形成离散的多蛋白质结构，其低聚状态和几何形状完全由金属配位控制。这种方法不仅产生复杂的生物组装体，而且还产生了蛋白质-蛋白质界面内构建的新型生物中心。到目前为止，我们已经收集了四个超蛋白组装的晶体学数据集，使用主要为小分子晶体学设计的设置，这产生了有限的数据分辨率/质量。在这个提议中，我们的目标是利用可调谐同步辐射来获得我们结晶的多达10个组件的高分辨率结构，彻底建立金属配位几何形状，并明确确认其中包含的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（负责生物固氮的酶）的铁蛋白（MoFeP）中潜在的质子转移途径。MoFeP催化8电子/8质子还原二氮为氨。虽然MoFeP中的电子转移已经被很好地理解，但质子转移途径尚未建立。使用细胞色素c氧化酶成功的方法，

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