Mapping and controlling the segmental dynamic networks of Cyclophilin A as they r

描述（由申请人提供）：目前设计治疗性蛋白质抑制剂的合理的、基于结构的方法在很大程度上局限于针对蛋白质的单一低能量构象的活性位点。由于溶液中的蛋白质不断采样，结构上的异质构象和变构效应经常被证明会影响远离活性位点的功能，这对合理的药物设计有重要的改善

DESCRIPTION (provided by applicant): Current rational, structure based approaches to designing therapeutic protein inhibitors are, largely, limited to targeting the active site of a sigle low energy conformation of a protein. As proteins in solution are continually sampling a structurally heterogeneous range of conformations and allosteric effects have often been shown to influence function distant from the active site, significant improvement to rational drug design could be realized by advancing our understanding of what dynamic motions exist in proteins and how they relate to function. In humans, Cyclophilin A (CypA), a peptidyl-prolyl isomerase, is involved in a range of biological function, including chaperone activity, intercellular signaling, and cytokine signaling, as well as having roles, both cytosolic and extracellularly, in driving development, progression, metastasis, and chemoprotection in a number of cancers, including lung, breast, colorectal, prostate, and liver. This study aims to measure dynamics in CypA and to unravel how these motions communicate throughout the protein to control enzymatic function. More broadly, the proposed work will develop several novel methodologies for studying this crucial dynamics-function relationship, which will be applicable to many protein systems. For the proposed studies, nuclear magnetic resonance (NMR) spectroscopy relaxation experiments will provide atomic resolution measurement of dynamic motions across timescales from picoseconds to milliseconds, including, critically, quantitative measurement of exchange rates near the timescale of catalysis (?s-ms). Additionally, several biophysical techniques, including NMR, ITC, and CD will be used to functionally characterize proteins in vitro. Two novel approaches will then be carried out to probe the dynamics-function relationship in CypA. In the first, residues, distal from the protein active site, will be identified for which dynamics are eiter altered by other CypA mutants or altered during catalysis (with of saturating concentrations of a model substrate), indicating that they are still within the functionally relevant networks of dynamic communication. Single site mutations of these residues will permit alteration of the global dynamics of the protein independent of structural changes to the active site and analysis of the functional implications of given dynamic changes. Multiple 'dynamic mutants', when analyzed in parallel, will reveal the networks through which regions of the protein communicate, as well as the motions involved in regulating function. The second approach will utilize recent advances in NMR data-driven molecular dynamics (MD) simulations to generate conformationally broad ensembles of CypA alone, during catalysis, and with dynamics altering mutations. A novel analytical technique, which allows for identification of partially correlated localization within these MD ensembles, will allow us to map, at high resolution, the specific pathways of communication in CypA. Combined, these complementary approaches will bring us closer to understanding how variable dynamics are communicated and how they relate to function, a critical step in advancing structure/dynamics based drug design.