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

结合基于化学位移和实验的方法来研究酶动力学

• 批准号: 9261551
• 负责人: ELAN Z EISENMESSER
• 金额: $ 29.24万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9261551
• 关键词: Active Sites; Address; Biochemical Reaction; Catalysis; Chemicals; Clinical; Collection; Coupled; Coupling; Cyclophilin A; Cyclophilins; Data; Development; Discipline; Disease; Distal; Engineering; Enzymes; Event; Goals; Human; Individual; Infection; Inflammatory; Lead; Malignant Neoplasms; Methodology; Methods; Molecular Conformation; Motion; Movement; Mutate; Mutation; Nuclear Magnetic Resonance; Peptidylprolyl Isomerase; Process; Protein Dynamics; Protein Region; Proteins; Publishing; Reaction; Relaxation; Relaxation Techniques; Resolution; Side; Signal Transduction; Site; Structure; Substrate Specificity; System; Techniques; Therapeutic; Translating; Work; base; computer studies; design; enzyme substrate; experimental study; flexibility; innovation; insight; macromolecule; millisecond; novel; pathogen; public health relevance; therapeutic target

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.

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。对于含有亲环蛋白a的酶，广泛接受的观点是，固有的构象交换包括一个“微调”以匹配催化功能的单一过程。然而，我们最近发表的关于亲环蛋白a的研究通过确定在没有底物的情况下活性位点内部和周围的几种不同的构象交换过程，对这一观点提出了挑战。此外，我们在本提案中提出的初步研究还表明，在催化过程中，多种构象交换过程是酶和底物的基础。这将为酶提供一个新的范例，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，而是事件的集合，或“动态片段”，正如我们的数据所表明的那样。本提案的第一个目标是开发、应用和验证旨在确定游离亲环蛋白a内以及周转期间存在的构象变化的计算和实验方法（aim 1的目标）。之后，我们将确定亲环素a活性位点远端的动态片段如何耦合以发挥功能，并确定这种耦合是否可以合理地设计以调节功能，正如我们最近对无底物亲环素a的研究所表明的那样（Aim 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发展与计算方法相结合，以探测活性亲环蛋白a在周转过程中的内在动力学。这样的研究将提供对酶的动力学如何与催化耦合的详细理解，并提供对酶的固有运动如何为催化功能做好准备的见解。考虑到亲环蛋白a占细胞总蛋白的近0.6%，并参与多种疾病（如癌症和炎症疾病）中上调的信号转导级联反应，了解这种重要酶的原子解析细节将具有更广泛的意义。

项目成果

Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding.

• DOI: 10.1016/j.jmb.2018.06.015
• 发表时间: 2018-09-14
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Paukovich N;Xue M;Elder JR;Redzic JS;Blue A;Pike H;Miller BG;Pitts TM;Pollock DD;Hansen K;D'Alessandro A;Eisenmesser EZ
• 通讯作者: Eisenmesser EZ