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，并为阐明序列特异性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：(i)三种密切相关的真菌性DNA弯曲蛋白，它们参与DNA包装和基因调控，使DNA发生近180度的弯曲：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伯氏疏螺旋体的Hbb；（ii）一种DNA修复蛋白MutS，它识别并结合不匹配的DNA位点，并使DNA弯曲约60度，从而启动DNA修复机制。这些蛋白质主要通过间接读出机制识别它们的结合位点，其中DNA的序列依赖性结构和灵活性/弯曲性起关键作用。将使用~10纳秒的激光温度跳变（T-jump）来扰动蛋白质-DNA复合物，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，蛋白质构象变化对t跃迁的响应将通过色氨酸荧光监测固有或引入的色氨酸残基的变化。单分子FRET测量也将在固定的DNA底物上进行，以探测弯曲构象在复合物中的分布，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DNA弯曲/不弯曲的动力学来探测DNA柔韧性/可弯曲性在识别机制中的作用，这种动力学适用于插入畸变的一系列底物，如错配或单t插入，这些底物与IHF/HU家族中的蛋白质具有广泛不同的亲和力；（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/不弯曲步骤同时发生，还是在一个不同的动力学步骤中发生；（iii）通过研究干扰特定蛋白质-DNA相互作用的突变对DNA弯曲/不弯曲率的影响，探索沿着反应坐标形成复合物的过渡态的性质；（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对错配的识别以及随后的DNA修复机制中atp酶驱动的步骤。该项目的一个新颖方面是应用激光t跳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。这项工作的更广泛的影响在于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，以更深入地了解其潜在机制。主要的教育目标是在ucic建立一个本科生物物理学专业，这将提供一个具有强大分析成分的多学科教育。本项目由分子与细胞生物科学部分子生物物理学和物理部生物物理项目共同支持。

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.