Jim Huntley, PhD National Center for Genome Resources jjh@ncgr.org Phone:(505) 995-4416

Title: NMR Investigation of Human CksHs1

Project Description:

The broad, long-term objective of this application is to evaluate the role of polypeptide-chain dynamics in the biomolecular pathways of cell-cycle control. The proposal's specific aim is to explore and characterize dynamics of a protein purported to have a central role in cell-cycle regulation. The protein, the cyclin-dependent kinase associated binding protein, CksHs1, has been shown to be critical for optimal degradation of the cyclin-dependent kinase inhibitor p27^Kip1. Because either mislocalization of p27^Kip1 and/or enhanced rates of p27^Kip1 degradation have recently been shown to be important contributory pathways of carcinogenesis, investigation of the mechanisms of CksHs1-mediated p27-degradation will improve our understanding of the normal processes that constitute cell growth. Previous studies have suggested that conformational flexibility of CksHs1 is an important determinant in its function. However, these investigations have not adequately characterized the nature of this flexibility or how it relates to CksHs1 function. Therefore, this investigation will characterize the polypeptide-chain dynamics of CksHs1 by use of NMR relaxation techniques to determine both the global and internal motions inherent in CksHs1 structure. CksHs1 backbone-amide NMR resonance assignment of ¹⁵N/¹³C-labeled protein will be accomplished by use of advanced three-dimensional NMR methodologies. Subsequent analysis of NMR ¹⁵ N-labeled CksHs1 in its free, monomeric form will then provide estimates of the extent of motions present in the protein backbone and timescales over which those motions take place. Identical methodologies will be employed to characterize the changes that occur to ¹⁵N-labeled CksHs1 dynamics upon binding of unlabeled CDK2. This investigation will then allow for a more complete and detailed picture of the role of CksHs1 dynamics in the p27-dependent pathway of CDK control.