Probing substrate/inhibitor binding to metalloenzymes using EPR

该奖项将资助迈阿密大学的Michael Crowder教授和David Tierney教授使用EPR(电子顺磁共振)波谱工具研究底物和抑制剂与金属酶的结合。曾经被认为是刚性分子，现在人们理解了酶--可以说是自然界最重要的生物分子--是动态的，酶的运动对催化是必不可少的。这项提议描述了当酶催化化学反应时探测酶运动的新方法；这些运动发生在不到千分之一秒的时间尺度上。该项目开发的技术原则上可以应用于所有酶，收集到的信息可能用于改变现有的酶以催化不同的反应。该项目将允许对大量本科生和研究生进行分子生物学、酶学和光谱学方面的培训，并将其整合到一个为小学生提供实践STEM活动的推广计划中。该研究项目将评估推广使用基于EPR的技术来确定在其他无法访问的系统/状态中的自旋间距离的新方法。这些技术被设计用来访问在室温下跨越微秒到毫秒的广泛过程，从亚单位间的运动到相对于衬底和活性中心金属离子的非结构化回路的运动。最终，该方法将被用来确定协同运动如何跟踪重要的催化事件。具体地说，该项目涉及一些实验，以确定快速冷冻淬火(RFQ)的可靠性--EPR衍生的距离变化伴随着金属-β-内酰胺酶NDM-1的一系列顺磁类似物的底物结合和周转，在大约100微秒到500毫秒以上的反应时间内冷冻淬火。将在X频段和Q频段进行平行研究，以确保内部一致性，并保证接触互动。还将进行实验，以检查可从CW-EPR和Deer产生可检测和可解释的偶极耦合的自旋物种组合的范围，以及最适合ESEEM询问的几个独特组合。这一目标将通过检查一组单和双自旋标记蛋白质中的可用排列来实现，这些蛋白质选择性地在双核活性中心的两侧负载抗磁性的锌(II)/镉(II)或顺磁性的Co(II)，以及自旋标记的底物/抑制物类似物，包括几个三标记物种。

With this award, the Chemistry of Life Processes Program in the Division of Chemistry is funding Professors Michael Crowder and David Tierney of Miami University to study substrate and inhibitor binding to metalloenzymes using the tools of EPR (electron paramagnetic resonance) spectroscopy. Once thought to be rigid molecules, it is now understood that enzymes--arguably Nature's most important biological molecules--are dynamic and that enzyme motions are essential for catalysis. This proposal describes novel methods to probe enzyme motions as the enzymes catalyze chemical reactions; these motions occur on timescales less than one one-thousandth of a second. The techniques developed in this project can, in principle, be applied to all enzymes, and the information gleaned can possibly be used to alter existing enzymes to catalyze different reactions. This project will allow for the training of a large number of undergraduate and graduate students in molecular biology, enzymology, and spectroscopy and be integrated into an outreach program that provides hands-on STEM activities for elementary school students.This research project will assess new ways to generalize the use of EPR-based techniques to determine inter-spin distances in otherwise inaccessible systems/states. The techniques are designed to access a wide range of processes that span microseconds to milliseconds at room temperature, from inter-subunit motions to the motion of unstructured loops relative to the substrate and active site metal ions. Ultimately, the method is to be used to determine how concerted motions track with important catalytic events. Specifically, the project involves experiments that determine the reliability of rapid-freeze quench (RFQ)-EPR-derived distance changes that accompany substrate binding and turnover in a series of paramagnetic analogs of the metallo-beta-lactamase NDM-1, freeze-quenched at reaction times ranging from around 100 microseconds to greater than 500 milliseconds. Parallel studies at both X- and Q-band will be conducted to ensure internal consistency and to guarantee access to the interactions. Experiments will also be conducted to examine the scope of spin-bearing species combinations that yield detectable, and interpretable, dipolar couplings from cw-EPR and DEER, and several unique combinations that are best suited for ESEEM interrogation. This goal will be accomplished by examining available permutations within a set of singly- and doubly spin-labeled proteins, selectively loaded with diamagnetic Zn(II)/Cd(II) or paramagnetic Co(II) in either side of the dinuclear active site, and spin-labeled substrate/inhibitor analogs, including several triply-labeled species.