Energetics of Protein-DNA Binding and Bending

蛋白质-DNA 结合和弯曲的能量学

• 批准号: 7149072
• 负责人: JOHN W SHRIVER
• 金额: $ 38.37万
• 依托单位: UNIVERSITY OF ALABAMA IN HUNTSVILLE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7149072
• 关键词: Archaea; DNA; DNA binding protein; X ray crystallography; bacterial proteins; binding sites; calorimetry; chemical stability; chromatin; circular dichroism; hydropathy; intermolecular interaction; ionic bond; model design /development; nuclear magnetic resonance spectroscopy; nucleic acid structure; osmotic pressure; physical model; protein denaturation; protein folding; protein structure function; site directed mutagenesis; structural biology; temperature; thermodynamics; thermophilic organism

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.

描述（由申请人提供）：蛋白质诱导的DNA失真对于许多正常细胞功能，包括基因调节，表达，重组和染色质组织的各个方面。 DNA失真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。对蛋白质引起的失真的能量学的定量理解落后于当前可用的结构信息水平。在很大程度上，这源于控制DNA失真并弯曲为实验变量的困难，在复合物中，可以获得量热和结构数据。高3个可SAC7D-DNA复合物非常适合用作模型基准系统，用于定量研究DNA弯曲的能量和由于较小的凹槽结合而引起的。在此提案中，我们集中于以下事实：可以实验操纵SAC7D-DNA复合物中的DNA失真水平，其中可以遵循结构变化和能量学。目的是定义溶液中SAC7D在DNA中诱导的失真，并将失真的直接测量与蛋白质-DNA相互作用的能量相关联。我们将使用荧光共振能量转移，DNA环化，NMR和量热法来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的失真和结合能的影响。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变形的大小以及结合和弯曲的相关能量。这将提供第一个直接的结构和量热测量，以DNA失真与蛋白质-DNA结合的能量的连接。这是一个基础研究项目，它将描述在许多与疾病相关的蛋白DNA复合物中发生的重要类型的DNA相互作用的能量学。 SAC7D是一种染色体域，在真核核蛋白中常见，也可以在HIV-1积分酶的DNA结合域中发现。该蛋白质通过与具有直接生物医学相关性的蛋白质相似的机制与DNA结合。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，这是药理学和治疗剂中潜在靶标。