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

通过顺磁金属 ESR 距离测量确定蛋白质 DNA 特异性决定因素

• 批准号: 1157712
• 负责人: Sunil Saxena
• 金额: $ 83.3万
• 依托单位: University of Pittsburgh
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1157712&HistoricalAwards=false
• 关键词: Protein; DNA; Specificity; Determinants; Paramagnetic

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.

智力优势：该项目取得了两项主要成就：（a）开发铜基脉冲ESR实验，目的是将该技术推广到通常不与金属结合的蛋白质，通过双量子相干（DQC）实现高达三倍的信噪比增强顺磁性金属离子的方法，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包装。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。一系列的距离限制铜（II）中心之间的位点选择性标记的蛋白质，X射线结构和分子建模将提供详细的了解包装和灵活性的侧链用于放置顺磁性金属离子在蛋白质中的选定位点。 这些信息对于NMR中基于顺磁弛豫增强的结构测定将是特别重要的。(b)探讨限制性内切酶EcoR Ⅰ的结构、蛋白质动力学和热力学之间的关系，提出蛋白质-DNA位点特异性识别的一般原理。脉冲ESR距离方法（DEER和DQC）和X-波段和W-波段连续波ESR光谱将被应用于研究EcoRI内切酶结合特异性，错配（一个不正确的碱基对）和非特异性DNA位点的复合物之间的动力学和距离分布的差异。一个新收购的W波段ESR光谱仪大大提高了区分蛋白质骨架动力学的自旋标签的运动的能力，并提供了令人兴奋的机会，以表征运动的亚纳秒时间尺度。 这将是第一次检查如何ESR为基础的动态参数不同，不仅在不同的位置在一个大分子复合物，而且在相应的位置在一组相关的识别复合物。这种比较测量将提供有关结构和动力学的微观差异如何导致热力学参数的宏观差异的信息。更广泛的影响：从事该项目的六到八名本科研究人员和两到三名研究生将接受化学和生物物理学之间的跨学科界面培训。 PI将通过在这些研究所积极开展研究，寻求从4年制大学招收本科生。将在匹兹堡地区为高中教师举办讲习班，使他们了解这项研究，并提供用于高中课程的单元。 高中学生的研讨会将使他们接触化学职业。 将分发从金属基电子自旋共振数据中提取距离的软件。 作为该项目支持的一部分，两名女性将接受ESR培训。在该项目中开发的研究思路和主题将用于现代化的本科物理化学课程。这一举措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。