POLYPEPTIDE-CHAIN DYNAMICS IN REGULATION OF CDK2

本申请的广泛的长期目标是评估多肽链动力学在细胞周期控制的生物分子途径中的作用。该提案的具体目标是探索和表征据称在细胞周期调控中起核心作用的蛋白质的结构动力学。蛋白质，细胞周期蛋白依赖性激酶相关的结合蛋白，CksHs 1，已被证明是至关重要的细胞周期蛋白依赖性激酶抑制剂p27（Kip 1）的最佳降解。由于p27（Kip 1）的错误定位和/或p27（Kip 1）降解速率的增加最近被证明是致癌的重要贡献途径，因此对CksHs 1介导的p27降解机制的研究将有助于进一步了解CksHs 1介导的p27降解的机制。 提高我们对构成细胞生长的正常过程的理解。以往的研究表明，构象灵活性的CksHs 1是一个重要的决定因素，在其功能。然而，这些调查没有充分的特点，这种灵活性的性质，或它如何涉及到CksHs 1功能。因此，本研究将通过使用NMR弛豫技术来确定CksHs 1结构中固有的全局和内部运动来表征CksHs 1的多肽链动力学。将通过使用先进的三维NMR方法完成15 N/3C-tabeld蛋白的CksHs 1骨架-酰胺NMR共振归属。随后分析的NMR弛豫参数为15 N-标记的CksHs 1在其自由的，单体的形式，然后将提供估计的运动的程度存在于蛋白质骨架和这些运动发生的时间尺度。 将采用相同的方法来表征未标记的CDK 2结合后15 N-标记的CksHs 1动力学发生的变化。这项调查，然后允许一个更完整和详细的图片中的作用CksHs 1动态的p27依赖性途径的CDK控制。

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.