Our long-term goal is to go beyond simply identifying motions within enzymes in order to characterize the structural changes themselves and address how dynamics within the micro-millisecond timescales are coupled to function. For enzymes that include cyclophilin-A, the widely accepted view is that an inherent conformational exchange comprises a single process that is "fine-tuned" to match the catalytic function. However, our recently published studies of cyclophilin-A have challenged this by identifying several distinct conformational exchange processes within and around the active site in the absence of substrate. Furthermore, our preliminary studies presented in this proposal also indicate that multiple conformational exchange processes underlie both enzyme and a substrate during catalysis. This would present a new paradigm for enzymes in which catalysis can no longer be viewed as a single conformational exchange event on the micro-millisecond timescale but instead a collection of events, or "dynamic segments", as our data indicates. Our first goal of this proposal is to develop, apply, and validate computational and experimental approaches that aim to identify the conformational changes present within free cyclophilin-A as well as during turnover (the goal of Aim 1). Afterwards, we will determine how dynamic segments distal to the cyclophilin-A active site are coupled to function and determine whether such coupling can be rationally engineered to modulate function as our recent work on substrate-free cyclophilin-A indicates (the goal of Aim 2). The novelty in our approach is that we combine developments in both NMR dynamics and NMR structure studies with computational approaches to probe active cyclophilin-A during turnover as well as the inherent dynamics of cyclophilin-A alone. Such studies will provide a detailed understanding of how the dynamics of an enzyme are coupled to catalysis and provide insight as to how the inherent motions of an enzyme are poised for catalytic function. Considering that cyclophilin-A comprises nearly 0.6% of total cellular protein and is involved in numerous signal transduction cascades upregulated during multiple diseases such as cancer and inflammatory disorders, understanding the atomic resolution details of such an important enzyme will have wider implications.
In addition to sequence and structure, protein dynamics are important for function and particularly critical for enzyme function that relies on conformational flexibility and rearrangements. While advances in spectroscopic techniques such as nuclear magnetic resonance (NMR) have revealed that the dynamics of many proteins comprise multiple dynamic regions, called dynamic segments, explicitly how these segments move and how they are coupled to function remains poorly understood. To this end, we will provide a physical picture of these dynamic segments within the enzyme cyclophilin-A by developing chemical shift-based structural methods and then alter the individual dynamic segments to both verify and determine their effects on function. The development and applications here of innovative methods that view active enzymes may then also prove to be of general importance for probing other processes at atomic resolution.
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