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.
Drug design, based on the physical structure of medically relevant proteins, faces a significant hurdle in that most current methods ignore the inherent internal dynamic motions of target proteins. These motions have been shown to regulate a wide range of protein function, yet we still lack a basic understanding of what motions physically exist and how they control proteins. This research aims at revealing high resolution descriptions of the internal motions that exist and unraveling the complex connection between these dynamic motions and protein function, critical progress towards more effective rational development of therapeutics.
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