Probing Dynamics Within an Enzyme Family

智力价值：酶是一种蛋白质，必须经历复杂的运动才能催化其生物学反应。一个相对较新的发现是，酶必须具有内在的灵活性，即使在没有底物的情况下，酶的运动也局限于其活性部位。在某种意义上，这些固有的运动已经为它们的催化功能预先编程，但这种关系到底是什么，它们如何跨越一系列酶仍然未知。虽然基因组学时代提供了序列和结构信息，但关于酶的动力学还知之甚少。这些知识将有助于理解决定蛋白质动力学的进化压力，以及决定序列和结构的进化压力。因此，本研究的重点是通过研究酶家族之间的序列、结构、动力学和功能之间的关系来了解酶和蛋白质是如何进化成动态运动的。目前的项目涉及对人类酶家族中与生物过程(微毫秒时间尺度)相同时间尺度上的动态行为进行比较的第一次调查。我们将利用最近发展起来的核磁共振技术来比较多个人类亲环素家族成员的动力学行为。这些酶可催化脯氨酸多肽键的可逆异构化，并发挥许多生物学作用，包括辅助蛋白质折叠、信号转导和蛋白质转运。这一反应的可逆性是本研究的一个关键方面，因为在周转过程中将直接探测活性酶复合体，并将直接确定它们与催化运动相关的内在运动之间的关系。此外，当与突变相结合时，将评估保守和非保守残基对整个酶家族的整体动态行为的特定部位的作用。因此，这些研究将首次确定是否存在导致酶内动态灵活性的交互作用的偏好，或者相反，限制运动的交互作用。更广泛的影响：这项研究将包括指导研究生和本科生，用一种根本不同的方法来表征大分子，超越静态的结构描述。该项目还包括旨在鼓励本科生，特别是代表人数不足的群体的成员从事生物学研究的活动。此外，对与生物过程相同时间尺度上的大分子运动的研究为直接研究广泛适用于其他生物体系的构象事件开辟了新的窗口。因此，激励年轻一代的研究人员开始在原子水平上提出一系列全新的问题是这项工作的一个基本方面。

Intellectual Merit: Enzymes are proteins that must undergo complex movements in order to catalyze their biological reactions. A relatively recent discovery is that enzymes must be inherently flexible and that enzymes undergo motions that are localized to their active sites even in the absence of substrates. These inherent movements have, in a sense, been pre-programmed for their catalytic function, but exactly what this relationship is and how they relate across a family of enzymes remains unknown. Although the genomics era has provided both sequence and structural information, little has been learned with regard to enzyme dynamics. Such knowledge will be instrumental in understanding the evolutionary pressures that dictate protein dynamics in addition to those that dictate both sequence and structure. Thus, the focus of this research is to understand how enzymes, and proteins in general, have evolved to undergo their dynamic movements by investigating the relationship between sequence, structure, dynamics, and function among a family of enzymes. The current project involves the first investigation comparing dynamic behavior on the same timescales as biological processes (micro-millisecond timescales) within a family of human enzymes. The dynamics behavior of multiple human cyclophilin family members will be compared using recently developed nuclear magnetic resonances (NMR) techniques. These enzymes catalyze the reversible isomerization of proline peptide bonds and play numerous biological roles, including aiding in protein folding, signal transduction, and protein trafficking. The reversible nature of this reaction is a critical aspect of this study, since the active enzyme complexes will be directly probed during turnover and the relationship between their inherent movements associated with catalytic motions will be directly determined. Moreover, when combined with mutagenesis, the site-specific roles of both conserved and non-conserved residues to the overall dynamic behavior across a family of enzymes will be assessed. Thus, these studies will determine for the first time whether there are preferences for interactions that lead to dynamic flexibility within enzymes or, conversely, interactions that limit motions. Broader impacts: This research will include the mentoring of both graduate and undergraduates students with a fundamentally different approach to characterizing macromolecules that looks beyond static structural descriptions. The project also includes activities aimed at stimulating undergraduates, especially members of underrepresented groups, to engage in biological research. Furthermore, the study of macromolecular motions on the same timescales as biological processes opens a new window to directly studying conformational events broadly applicable to other biological systems. Thus, inspiring a young generation of researchers to begin asking an entirely new set of questions at the atomic level is a fundamental aspect of this work.