Combining chemical shift-based and experimental approaches to study enzyme dynami

描述(由申请人提供)：我们的长期目标是超越简单地识别酶内的运动，以便表征结构变化本身，并解决微毫秒时间尺度内的动力学如何耦合到功能。对于包括亲环素-A的酶，普遍接受的观点是，固有的构象交换包括一个“微调”的单一过程，以匹配催化功能。然而，我们最近发表的关于亲环素-A的研究发现，在没有底物的情况下，活性中心内部和周围的几个不同的构象交换过程对此提出了挑战。此外，我们的初步研究还表明，在催化过程中，酶和底物之间存在多个构象交换过程。这将为酶提供一种新的范式，其中催化不再被视为微毫秒时间尺度上的单个构象交换事件，而是如我们的数据所示的事件的集合，或“动态片段”。我们这项提议的第一个目标是开发、应用和验证计算和实验方法，旨在确定游离亲环素-A以及在周转期间(目标1的目标)中存在的构象变化。然后，我们将确定亲环素-A活性部位远端的动态片段是如何与功能偶联的，并确定这种偶联是否可以被合理地设计来调节功能，正如我们最近关于无底物亲环素-A的工作所表明的那样(目标2的目标)。我们方法的新颖之处在于，我们将核磁共振动力学和核磁共振结构研究的进展与计算方法相结合，以探索亲环素-A在周转过程中的活性以及亲环素-A单独的内在动力学。这样的研究将提供对酶的动力学如何与催化相耦合的详细了解，并提供关于酶的内在运动如何准备用于催化功能的洞察。考虑到亲环素-A占细胞总蛋白的近0.6%，并参与多种疾病(如癌症和炎症性疾病)中上调的许多信号转导通路，了解这种重要酶的原子分辨率细节将具有更广泛的意义。

DESCRIPTION (provided by applicant): 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.