Protein DNA Specificity Determinants by Paramagnetic Metal Based ESR Distance Measurements

基于顺磁性金属的 ESR 距离测量的蛋白质 DNA 特异性决定因素

• 批准号: 0842956
• 负责人: Sunil Saxena
• 金额: $ 58.58万
• 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: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0842956&HistoricalAwards=false
• 关键词: Protein; DNA; Specificity; Determinants; Paramagnetic

This project will probe the linkage between molecular structure, molecular motions and function in proteins that recognize and associate specific sites on DNA, in order to deduce general principles of site-specific protein-DNA recognition. Past research using EcoRI endonuclease as a model has suggested that when a protein binds to its "correct" site on DNA, both the protein and the DNA suffer losses of conformational-vibrational mobility, but that these changes are much less pronounced when the protein binds at an incorrect DNA site. If verified, this would provide deeper insight into how a protein chooses among DNA sites. This hypothesis cannot be readily tested since the incorrect protein-DNA complexes are presently inaccessible to crystallography or nuclear magnetic resonance methods in solution. Electron spin resonance (ESR) spectroscopy provides an exciting alternative route to assessing structural and dynamic differences between the various classes of complexes. Intellectual Merit: Early results on a DNA-protein complex by pulsed ESR establish an entirely new methodology that can measure the solution structure and range of conformational states for complexes with different classes of DNA sites. Such comparative measurements can provide information on how microscopic differences in molecular motions and structure contribute to the macroscopic differences in affinity and catalytic rates. In order to better quantify differences among the complexes, ESR methods that exploit bound copper(II) ions and site-directed nitroxide spin labeling will be developed, and applied to EcoRI-DNA complexes. Preliminary evidence shows that copper(II) coordinates exclusively between His114 and a DNA phosphate in the EcoRI-DNA complex, thus providing a unique reference point. Copper(II) coordination prevents significant motion of the ion, so that the resulting ESR signals aid in deconvoluting protein flexibility from motions of the spin label. The specific scientific aims are to: (1) Establish a pulsed electron spin resonance (ESR) method to measure large metal-metal and metal-nitroxide distances in proteins. (2) Leverage metal-spin label measurements to determine point-to-point distances and the range of conformational states of the endonuclease in specific, miscognate and nonspecific DNA complexes. (3) Combine research with outreach to 4-year colleges, local high schools and undergraduates. Broader Impact: Two to three undergraduate researchers and 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. Research ideas and themes developed in the project will be used to modernize the undergraduate Physical Chemistry laboratory. 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 curriculum without the creation of new course work. An annual workshop will be organized for high-school teachers in the Pittsburgh area to orient them with this research. Software for the extraction of distances from metal-based ESR data will be disseminated. This grant supports ESR training for females.

本项目将探索蛋白质中识别和结合DNA上特定位点的分子结构、分子运动和功能之间的联系，以推导出位点特异性蛋白质-DNA识别的一般原理。过去使用EcoRI内切酶作为模型的研究表明，当蛋白质结合到DNA上的“正确”位置时，蛋白质和DNA都会失去构象-振动迁移率，但当蛋白质结合在错误的DNA位置时，这些变化就不那么明显了。如果得到证实，这将为蛋白质如何在DNA位点中进行选择提供更深入的了解。这一假设不容易被检验，因为错误的蛋白质-DNA复合体目前不能用结晶学或核磁共振方法在溶液中获得。电子自旋共振(ESR)谱为评估不同类型络合物之间的结构和动力学差异提供了一种令人兴奋的替代方法。智慧价值：脉冲ESR对DNA-蛋白质复合体的早期结果建立了一种全新的方法，可以测量具有不同类别DNA位点的复合体的溶液结构和构象状态范围。这种比较测量可以提供关于分子运动和结构的微观差异如何导致亲和力和催化速率的宏观差异的信息。为了更好地量化配合物之间的差异，将发展利用结合铜(II)离子和定点氮氧化物自旋标记的ESR方法，并将其应用于EcoRI-DNA配合物。初步证据表明，铜(II)仅与EcoRI-DNA复合体中的His114和DNA磷酸之间配位，从而提供了一个独特的参照点。铜(II)的配位阻止了离子的显著运动，因此产生的ESR信号有助于使蛋白质的灵活性从自旋标记的运动中解卷。具体的科学目标是：(1)建立一种脉冲电子自旋共振(ESR)方法来测量蛋白质中大的金属-金属和金属-氮氧化物的距离。(2)利用金属自旋标记测量来确定特定、异源和非特定DNA复合体中核酸内切酶的点到点距离和构象范围。(3)将研究与对四年制大学、当地高中和本科生的推广相结合。更广泛的影响：在这个项目上工作的两到三名本科生研究人员和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。该项目中提出的研究思路和主题将用于本科生物理化学实验室的现代化。这一倡议符合美国化学学会的呼吁，即在不创建新的课程工作的情况下，将化学研究的跨学科和综合趋势纳入本科课程。将为匹兹堡地区的高中教师组织一年一度的研讨会，以指导他们进行这项研究。将分发从基于金属的ESR数据中提取距离的软件。这笔赠款支持为女性提供ESR培训。