The proposed research will be conducted by Dr. Carlos Ramos, primarily in Campinas, Brazil, at the Brazilian Synchrotron Laboratory (LNLS), in collaboration with Dr. Douglas Cyr of the Department of Cell Biolog at UNC-Chapel Hill. The parent grant held by Dr. Cyr GM R01 NIH R01GM56981 is focused on understanding the mechanisms by which Hsp40 proteins regulate Hsp70 action in cell stress. This FIRCA award seeks to extend the aims of the parent grant and previous work of Dr. Ramos to study the mechanisms by which Type I and Type II Hsp40s specify the cellular functions of Hsp70. One goal of this proposal is to determine the structural elements that control the quaternary structure of Type I and Type II Hsp40s. Dr. Cyr has demonstrated that Type I and Type II Hsp40s exhibit differences in substrate specificity and protein folding activity that control the cellular function of Hsp70. However, the mechanism for Hsp40 action as a protein folding factor is unknown. Dr. Ramos has demonstrated that Type I and Type II Hsp40 proteins and have grossly different quaternary structures, which may account for the functional differences exhibited by these co-chaperones. In this aims we set of defined to experiments to define the features that control the quaternary structure. To accomplish this goal the Ramos laboratory will utilize small angle X-ray scattering, cross-linking and analytic ultracentrifugation to study the quaternary structures of Hsp40s. At present we have high resolution structural information on fragments of Hsp70 and Hsp40, but there is little information that describes contacts formed between Hsp70 and Hsp40 during the process of protein folding. Since the field has not have been able to obtain high resolution structural data to describe Hsp70/Hsp40 interactions, as a second goal we propose to utilize SASX and cross-linking/mass spectroscopy approaches to build such models. Then we will test the predictions made from these models by designing mutants and testing there activity in vivo and in vitro functional assays. These data will determine the mechanism by which Type I and Type II Hsp40s interact with Hsp70 to suppress protein aggregation and facilitate protein folding/assembly. ? ?
