The broad, long-term objective of this application is to evaluate the role of polypeptide-chain dynamics in the biomolecutar pathways of cell-cycle control. The proposal's specific aim is to explore and characterize the structural 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/3C-tabeld protein will be accomplished by use of advanced three-dimensional NMR methodologies. Subsequent analysis of NMR relaxation parameters for 15N-labeld 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-labeld 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.
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