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

本研究的总体目标是探索蛋白质和DNA在复合体形成过程中的分子重排动力学，并为阐明序列和结构特异性的分子起源提供物理基础。这个项目将专注于两类蛋白质：(I)三种密切相关的真细菌DNA弯曲蛋白，参与DNA包装和基因调控，使DNA大幅弯曲近180度：大肠杆菌整合宿主因子(IHF)，来自Anabeana的组蛋白样蛋白(Ahu)，以及来自导致伯氏疏螺旋体的莱姆病的Hbb；以及(Ii)DNA修复蛋白MutS，它识别并错配结合到DNA位点，使DNA弯曲约60度，从而启动DNA修复机制。这些蛋白质主要通过一种间接读出机制识别它们的结合部位，其中DNA的序列依赖结构和柔韧性起着关键作用。10纳秒激光温度跳跃(T-JUMP)将被用来扰动蛋白质-DNA复合体，结合的DNA底物的弯曲/不弯曲动力学将通过末端标记的DNA底物上的时间分辨FRET来监测。此外，将通过固有的或引入的Trp残基的Trp荧光变化来监测响应T-JUMP的蛋白质构象的变化。还将在带有结合蛋白的固定化DNA底物上进行单分子FRET测量，以探索弯曲构象在复合体中的分布，并在单分子水平上提供动力学信息。本研究的具体目的是：(I)通过测量一系列具有插入变形的底物(如错配或与IHF/HU家族蛋白质具有广泛不同亲和力的单T插入片段)的DNA弯曲/非弯曲动力学，来探讨DNA柔性/可弯曲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：蛋白质的构象变化是与DNA弯曲/非弯曲步骤同时发生的，还是以不同的动力学步骤发生的；(Iii)通过研究干扰特定蛋白质-DNA相互作用的突变对DNA弯曲/不弯曲速率的影响，探索沿着复杂反应坐标的过渡态的性质；(Iv)探索与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修复机制中随后由ATPase驱动的步骤。该项目的一个新方面是应用激光T跳跃技术以亚微秒的时间分辨率探测蛋白质-DNA相互作用的动力学。这项工作的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，包括其他调节和DNA修复蛋白质，以更深入地了解潜在的机制。主要的教育目标是在UIC建立一个生物物理学本科专业，这将提供一个多学科的教育，具有很强的分析成分。该项目由分子和细胞生物科学部的分子生物物理学和物理学部的生物物理计划共同支持。

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.