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.
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