Protein DNA Specificity Determinants by Paramagnetic Metal-Based ESR Distance Measurements

智力价值：与该项目相关的两项主要成就：(A)开发基于铜的脉冲ESR实验，目的是将该技术推广到正常情况下不结合金属的蛋白质，通过顺磁金属离子的双量子相干(DQC)方法实现信噪比最多提高三倍，并利用这种技术来了解通常用于自旋标记蛋白质的侧链的堆积。距离测量方法将对理解传统生物物理方法无法研究的蛋白质结构-功能关系的能力产生重大影响。选择性地标记在蛋白质上的铜(II)中心之间的一系列距离限制、X射线结构和分子建模将提供对用于将顺磁性金属离子放置在蛋白质中选定位置的侧链的堆积和灵活性的详细见解。这些信息对于在核磁共振中基于顺磁驰豫增强的结构确定特别重要。(B)探讨限制性内切酶EcoRI的结构、蛋白质动力学和热力学之间的关系，以引出位点特异性蛋白质-DNA识别的一般原理。我们将利用脉冲ESR距离法(DER和DQC)以及X波段和W波段连续波谱技术来研究EcoRI核酸内切酶结合于特定、异源(一个错误的碱基对)和非特定DNA位点的复合体之间的动力学和距离分布的差异。新获得的W波段ESR光谱仪极大地增强了区分蛋白质骨架动力学和自旋标记运动的能力，并提供了在亚纳秒时间尺度上表征运动的令人兴奋的机会。这将是关于基于ESR的动力学参数不仅在大分子复合体中的不同位置，而且在一组相关识别复合体中的相应位置如何不同的第一次检查之一。这种比较测量将提供有关结构和动力学的微观差异如何影响热力学参数的宏观差异的信息。更广泛的影响：参与该项目的6至8名本科生研究人员和2至3名研究生将在化学和生物物理学之间的跨学科界面上接受培训。PI将通过在四年制学院积极介绍研究成果来招收本科生。将在匹兹堡地区为高中教师组织讲习班，以指导他们进行这项研究，并提供高中课程中使用的模块。为高中生开设的工作坊将使他们接触到化学职业。将分发从基于金属的ESR数据中提取距离的软件。作为该项目支持的一部分，两名妇女将接受ESR培训。该项目中开发的研究思路和主题将用于本科物理化学课程的现代化。这一倡议符合美国化学学会的呼吁，即在不创建新课程的情况下，将化学研究的跨学科和综合趋势纳入本科课程。

Intellectual Merit: There are two major accomplishments associated with this project: (a) Developing copper-based pulsed ESR experiments with the aim of generalizing the technique to proteins that do not normally bind metals, achieving up to a three-fold enhancement in signal-to-noise ratio through double quantum coherence (DQC) methods for paramagnetic metal ions and exploiting such techniques to learn about the packing of the side chain that is typically used to spin label proteins. The distance measurement methodology will have a significant impact on the ability to understand structure-function relationships in proteins that cannot be studied by traditional biophysical methods. A series of distance constraints between copper(II) centers site-selectively labeled on a protein, X-ray structures and molecular modeling will provide detailed insights into the packing and flexibility of side chains used to place paramagnetic metal ions at selected sites in proteins. Such information will be especially important for paramagnetic relaxation enhancement based structure determinations in NMR. (b) Probe the relationships among structure, protein dynamics, and thermodynamics in the restriction endonuclease EcoRI to adduce general principles of site-specific protein-DNA recognition. Pulse-ESR distance methods (both DEER and DQC) and X-band and W-band continuous wave ESR spectroscopy will be applied to study the differences in dynamics and distance distributions between complexes of EcoRI endonuclease bound to specific, miscognate (one incorrect base pair) and nonspecific DNA sites. A newly acquired W-band ESR spectrometer greatly enhances the ability to distinguish protein backbone dynamics from motions of the spin label and provides the exciting opportunity to characterize motions on the sub-nanosecond timescale. This will be one of the first examinations of how ESR-based dynamic parameters differ not only at different locations within a macromolecular complex, but also at corresponding locations in a set of related recognition complexes. Such comparative measurements will provide information on how microscopic differences in structure and dynamics contribute to macroscopic differences in thermodynamic parameters. Broader Impact:Six to eight undergraduate researchers and two to three graduate students working on this project will be trained at the interdisciplinary interface between chemistry and biophysics. The PI will seek to recruit undergraduates from 4-year colleges by actively presenting research at those institutes. Workshops will be organized for high-school teachers, in the Pittsburgh area, to orient them with this research as well as provide modules for use in high school curricula. Workshops for high school students will expose them to careers in Chemistry. Software for the extraction of distances from metal-based ESR data will be disseminated. Two women will be trained in ESR as part of the support from this project. Research ideas and themes developed in the project will be used to modernize the undergraduate Physical Chemistry course. This initiative is in line with the American Chemical Society's call for the incorporation of interdisciplinary and integrative trends of chemical research into undergraduate curricula without the creation of new course work.