Study of the Dynamics of Protein-DNA Interactions to Probe Site-Specific Recognition

蛋白质-DNA 相互作用动力学研究以探测位点特异性识别

• 批准号: 0721937
• 负责人: Anjum Ansari
• 金额: $ 92万
• 依托单位: University of Illinois at Chicago
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0721937&HistoricalAwards=false
• 关键词: Study; Dynamics; Protein; DNA; Interactions

The overall goals of this study are to probe the dynamics of molecular rearrangements in both the protein and the DNA during complex formation and to provide a physical basis for elucidating the molecular origins of sequence- and structure-specificity. This project will focus on two classes of proteins: (i) three closely related eubacterial DNA-bending proteins involved in DNA packaging and gene regulation that dramatically bend the DNA, by nearly 180 degrees: E. coli Integration Host Factor (IHF), histone-like protein from Anabeana (AHU), and Hbb from the Lyme-disease causing spirochete Borrelia burgdorferi; and (ii) a DNA-repair protein, MutS, that recognizes and binds to DNA sites with a mismatch, and bends the DNA by about 60 degrees, thus initiating the DNA repair machinery. These proteins recognize their binding sites primarily by an indirect readout mechanism, in which the sequence-dependent structure and flexibility/bendablity of the DNA play a key role. A ~10 nanosecond laser temperature-jump (T-jump) will be used to perturb the protein-DNA complex, and the bending/unbending dynamics of the bound DNA substrate will be monitored with time-resolved FRET on end-labeled DNA substrates. In addition, protein conformational changes in response to the T-jump will be monitored with Trp fluorescence changes of intrinsic or introduced Trp residues. Single-molecule FRET measurements will also be carried out on immobilized DNA substrates with bound protein to probe the distribution of bent conformations in the complex, and to provide dynamics information at the single-molecule level. The specific aims of this study are to (i) probe the role of DNA flexibility/bendability in the recognition mechanism by measuring the kinetics of DNA bending/unbending for a range of substrates with inserted distortions such as mismatches or single-T insertions that bind with widely varying affinities to the proteins in the IHF/HU family; (ii) probe directly the conformational changes in the protein to address the question: do protein conformational changes occur concurrently with the DNA bending/unbending step or in a distinct kinetic step; (iii) probe the nature of the transition state along the reaction coordinate for complex formation by investigating the effect of mutations that perturb specific protein-DNA interactions on the DNA bending/unbending rates; (iv) probe the DNA bending kinetics in mismatched substrates bound to MutS to investigate how DNA bending dynamics influence mismatch recognition by MutS and subsequent ATPase-driven steps in the DNA repair mechanism.A novel aspect of this project is the application of laser T-jump techniques to probe the dynamics of protein-DNA interactions with submicrosecond time-resolution. The broader impact of this work is in the potential for extending these kinetics measurements to a wider class of protein-DNA systems, including other regulatory and DNA-repair proteins, for a deeper understanding of the underlying mechanisms. The primary educational goal is to establish an undergraduate Biophysics major at UIC, which will provide a multidisciplinary education with a strong analytical component. This project is being jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences and the Biological Physics Program in the Physics Division.

本研究的总体目标是探究复合物形成过程中蛋白质和 DNA 分子重排的动态，并为阐明序列和结构特异性的分子起源提供物理基础。 该项目将重点研究两类蛋白质：(i) 三种密切相关的真细菌 DNA 弯曲蛋白，涉及 DNA 包装和基因调控，可将 DNA 急剧弯曲近 180 度：大肠杆菌整合宿主因子 (IHF)、来自 Anabeana (AHU) 的组蛋白样蛋白和来自引起莱姆病的螺旋体伯氏疏螺旋体的 Hbb； (ii) DN​​A 修复蛋白 MutS，它识别并结合不匹配的 DNA 位点，并将 DNA 弯曲约 60 度，从而启动 DNA 修复机制。这些蛋白质主要通过间接读出机制识别其结合位点，其中 DNA 的序列依赖性结构和灵活性/可弯曲性起着关键作用。约 10 纳秒的激光温度跳跃 (T-jump) 将用于扰动蛋白质-DNA 复合物，并且将通过末端标记 DNA 底物上的时间分辨 FRET 来监测结合 DNA 底物的弯曲/不弯曲动态。此外，将通过内在或引入的色氨酸残基的色氨酸荧光变化来监测响应T跳跃的蛋白质构象变化。单分子 FRET 测量还将在带有结合蛋白的固定 DNA 基质上进行，以探测复合物中弯曲构象的分布，并提供单分子水平的动态信息。本研究的具体目的是 (i) 通过测量一系列具有插入扭曲（例如错配或单 T 插入）的底物的 DNA 弯曲/伸直动力学，来探讨 DNA 柔性/弯曲性在识别机制中的作用； (ii) 直接探测蛋白质的构象变化以解决以下问题：蛋白质构象变化是与DNA弯曲/伸直步骤同时发生还是在不同的动力学步骤中发生； (iii) 通过研究扰乱特定蛋白质-DNA 相互作用的突变对 DNA 弯曲/不弯曲速率的影响，探索复合物形成的反应坐标上过渡态的性质； (iv) 探测与 MutS 结合的错配底物中的 DNA 弯曲动力学，以研究 DNA 弯曲动力学如何影响 MutS 的错配识别以及 DNA 修复机制中后续 ATP 酶驱动的步骤。该项目的一个新颖方面是应用激光 T 跳跃技术以亚微秒时间分辨率探测蛋白质-DNA 相互作用的动力学。 这项工作更广泛的影响在于有可能将这些动力学测量扩展到更广泛的蛋白质-DNA 系统，包括其他调节蛋白和 DNA 修复蛋白，以便更深入地了解潜在机制。主要教育目标是在伊利诺伊大学芝加哥分校建立生物物理学本科专业，这将提供具有强大分析成分的多学科教育。 该项目得到了分子和细胞生物科学部的分子生物物理学和物理部的生物物理项目的共同支持。