Energetics of Protein-DNA Binding and Bending

描述（由申请人提供）：蛋白质诱导的DNA畸变对许多正常细胞功能至关重要，包括基因调控、表达、重组和染色质组织等方面。DNA畸变是一个能量昂贵的过程，与蛋白质-DNA复合物的亲和力和功能密切相关。对蛋白质诱导的扭曲的能量学的定量理解远远落后于目前可用的结构信息水平。在很大程度上，这源于控制DNA畸变和弯曲作为复合实验变量的难度，其中热量和结构数据都是可获得的。超热稳定的Sac7d-DNA复合物是唯一适合作为一个模型基准系统，用于定量研究DNA弯曲和解绕的能量学，由于轻微的凹槽结合。在本提案中，我们关注的事实是，可以通过实验操纵Sac7d-DNA复合物中的DNA扭曲水平，其中结构变化和能量学可以遵循。目的是定义Sac7d在溶液中诱导的DNA畸变，并将畸变的直接测量与蛋白质-DNA相互作用的能量学联系起来。我们将利用荧光共振能量转移、DNA环化、核磁共振和量热等方法系统地研究特定氨基酸残基的作用，以及DNA序列和长度对蛋白质诱导畸变和结合能的影响。此外，我们将研究细胞条件的影响，包括盐浓度、特定的反离子和渗透压，对扭曲程度和相关的结合和弯曲能量学的影响。这将提供DNA扭曲与蛋白质-DNA结合能量学联系的第一个直接结构和量热测量。这是一个基础研究项目，它将提供在许多疾病相关的蛋白质-DNA复合物中发生的一种重要类型的DNA相互作用的能量学描述。Sac7d是一个染色质结构域，是真核生物核蛋白中常见的折叠，也存在于HIV-1整合酶的dna结合结构域中。该蛋白通过类似于在具有直接生物医学相关性的蛋白质中观察到的机制与DNA结合。该结果将增强我们合理控制相关蛋白- dna结合相互作用的能力，这些相互作用是药理学和治疗学的潜在靶点。

DESCRIPTION (provided by applicant): Protein-induced DNA distortion is essential for many normal cellular functions, including aspects of gene regulation, expression, recombination, and chromatin organization. DNA distortion is an energetically costly process that is intimately linked to the affinity and function of the protein-DNA complexes. A quantitative understanding of the energetics of protein-induced distortion lags far behind the level of structural information currently available. To a large extent, this derives from the difficulty of controlling DNA distortion and bending as experimental variables in complexes where both calorimetric and structural data are obtainable. The hyper-thermostable Sac7d-DNA complex is uniquely suited to serve as a model benchmark system for quantitative studies of the energetics of DNA bending and unwinding due to minor groove binding. In this proposal, we focus on the fact that it is possible to experimentally manipulate the level of DNA distortion in Sac7d-DNA complexes where structural changes and energetics can be followed. The goal is to define the distortion induced in DNA by Sac7d in solution, and to correlate direct measures of distortion with the energetics of the protein-DNA interaction. We will use fluorescence resonance energy transfer, DNA cyclization, NMR, and calorimetry to systematically investigate the role of specific amino acid residues, as well as the influence of DNA sequence and length on protein-induced distortion and binding energetics. In addition, we will investigate the influence of cellular conditions, including salt concentration, specific counter-ions, and osmolarity, on the magnitude of distortion and the associated energetics of binding and bending. This will provide the first direct structural and calorimetric measure of the linkage of DNA distortion to the energetics of protein-DNA binding. This is a basic research project which will provide a description of the energetics of an important type of DNA interaction that occurs in many disease-related protein-DNA complexes. Sac7d is a chromo-domain, a fold common in eukaryotic nuclear proteins that is also found in the DNA-binding domain of HIV-1 integrase. The protein binds to DNA via mechanisms similar to those observed in proteins with direct biomedical relevance. The results will enhance our ability to rationally control related protein-DNA binding interactions that are potential targets in pharmacology and therapeutics.