DOMAIN SPECIFIC INTERACTIONS OF CENTRIN

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。中心蛋白是微管组织中心（MTOC）的重要组成部分，在有丝分裂过程中介导染色体分离。除了与其他MTOC组分相互作用外，已知其与在细胞周期和受损DNA修复中重要的蛋白质相互作用。中心蛋白C-末端结构域的多个靶标已经被确定，并且我们实验室已经表征了该结构域与其靶标之一结合的结构基础。已经提出了一个模型，其中中心蛋白的C-末端结构域作为一个锚靶蛋白在基础水平的钙和N-末端结构域作为传感器的钙信号。目标和N-末端结构域的结合模式仍有待鉴定和表征。我们假设该结构域具有与C-末端结构域不同的靶特异性和作用模式。我们研究的目标是确定N-末端结构域的结合伙伴，并使用结构方法来表征其与其特定靶标相互作用的方式。我们的具体合作目标是：1）确定中心蛋白N端结构域的结合伙伴。酵母双杂交筛选将用于鉴定潜在的蛋白质-蛋白质相互作用，使用S. cerevisiae，C. rheinhardii和H.在这些测定中，智人中心蛋白作为诱饵。2)测量相互作用蛋白质对中心蛋白的亲和力。将通过CD或荧光光谱法直接监测结合，并从这些数据计算中心蛋白结构域的相对亲和力。此外，将使用类似方法评估每种相互作用的钙依赖性。3)确定中心蛋白-靶复合物的结构。我们已经确定了中心蛋白的N-末端和C-末端结构域的结构，使用NMR光谱获得的数据。我们将利用核磁共振技术，以获得网站之间的相互作用中心蛋白和它的目标具体信息。将使用X射线晶体学或NMR确定与蛋白质靶标结合的中心蛋白的结构。

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. Centrin is an essential component of the microtubule organizing centers (MTOC), which mediates chromosome segregation during mitosis. In addition to interacting with other MTOC components, it is known to interact with proteins important in both cell cycling and repair of damaged DNA. Multiple targets for the C-terminal domain of centrin have been identified, and the structural basis for binding by this domain to one of its targets has been characterized by our laboratory. A model has been proposed in which the C-terminal domain of centrin serves as an anchor to target proteins at the basal level of calcium and the N-terminal domain serves as the sensor of calcium signals. Targets and the mode of binding for the N-terminal domain remain to be identified and characterized. We hypothesize that this domain has target specificity and mode of action that is distinct from the C-terminal domain. The goal of our research is to identify binding partners for the N-terminal domain, and, using structural methods, to characterize the way in which it interacts with its specific targets. Our specific aims for this collaboration are: 1) Identify binding partners for N-terminal domain of centrin. The Yeast-Two-Hybrid screen would be used to identify potential protein-protein interactions, using domain and full-length constructs for S. cerevisiae, C. rheinhardii and H. sapiens centrins as bait in these assays. 2) Measure the affinity of interacting proteins for centrin. Binding will be monitored directly by CD or fluorescence spectroscopy, and relative affinities for centrin domains calculated from these data. In addition, the calcium dependence of each interaction will be assessed, using similar methods. 3) Determine the structure of centrin-target complexes. We have determined structures of both the N-terminal and C-terminal domains of centrin, using data obtained by NMR spectroscopy. We will make use of NMR techniques to obtain site specific information for interactions between centrin and its targets. Structural determinations of centrin bound to protein targets will be made using X-ray crystallography or NMR.