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动作，以表征结构上的变化本身并解决微毫升时间表中的动态如何耦合到功能。对于包括环蛋白A的酶，广泛接受的观点是固有的构象交换包含一个“微调”以匹配催化功能的单个过程。但是，我们最近发表的关于环磷脂A的研究通过在没有底物的情况下确定了活跃部位和周围周围的几个不同构象交换过程提出了质疑。此外，该提案中提出的初步研究还表明，在催化过程中酶和底物的多个构象交换过程均为酶。这将为酶提供一个新的范式，其中催化不再被视为微毫秒次时尺度上的单个构象交换事件，而是如我们的数据所示，而是一系列事件或“动态片段”。 该提案的第一个目标是开发，应用和验证计算和实验方法，旨在确定自由环蛋白A和周转期间中存在的构象变化（AIM 1的目标）。之后，我们将确定如何耦合至周期素A活性位点的动态段，并确定是否可以合理地设计此类耦合，以调节我们最近无基质环磷脂A的工作（AIM 2的目标）。我们方法的新颖性是，我们将NMR动力学和NMR结构研究中的发展与计算方法相结合，以探测周转期间活跃的环磷脂-A，以及单独的环磷脂A的固有动力学。此类研究将详细了解酶的动力学如何与催化结合在一起，并就酶的固有运动如何促进催化功能提供见解。考虑到环糖素A占总细胞蛋白的近0.6％，并且参与多种疾病（例如癌症和炎症性疾病）中上调的许多信号转导级联，因此了解这种重要酶的原子分辨率细节将具有更广泛的影响。

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