Energetics of Protein-DNA Binding and Bending

蛋白质-DNA 结合和弯曲的能量学

• 批准号: 7255664
• 负责人: JOHN W SHRIVER
• 金额: $ 38.25万
• 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/7255664
• 关键词: Affinity; Amino Acids; Anions; Base Pairing; Basic Science; Benchmarking; Binding; Biological Models; Calorimetry; Cations; Cell physiology; Chromatin; Classification; Complex; Condition; Cyclization; DNA; DNA Binding; DNA Binding Domain; DNA Fingerprinting; DNA Minor Groove Binding; DNA Sequence; DNA-Binding Proteins; DNA-Protein Interaction; Data; Data Collection; Decompression Sickness; Dependence; Disease; Entropy; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Expression Regulation; Genetic Recombination; Goals; HIV-1 integrase; Human Development; Ions; Knowledge; Length; Link; Measures; Minor Groove; Modeling; Nuclear Protein; Nuclear Proteins; Numbers; Osmolar Concentration; Pharmacology; Play; Process; Property; Protein Binding; Proteins; Range; Research Personnel; Research Project Grants; Role; Side; Sodium Chloride; Solutions; Solvents; Structure; System; Temperature; Therapeutic; Water; Work; enthalpy; improved; intercalation; novel strategies; programs; protein function; protein structure

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 畸变对于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 畸变是一个耗能巨大的过程，与蛋白质-DNA 复合物的亲和力和功能密切相关。对蛋白质引起的变形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，这是由于在可以获得量热和结构数据的复合物中控制 DNA 扭曲和弯曲作为实验变量的难度。超热稳定的 Sac7d-DNA 复合物特别适合作为模型基准系统，用于定量研究由于小沟结合而引起的 DNA 弯曲和解旋能量学。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，从而跟踪结构变化和能量学。目标是定义 Sac7d 在溶液中引起的 DNA 畸变，并将畸变的直接测量与蛋白质-DNA 相互作用的能量关联起来。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扭曲和结合能量的影响。此外，我们将研究细胞条件（包括盐浓度、特定反离子和渗透压）对扭曲程度以及相关的结合和弯曲能量学的影响。这将为 DNA 畸变与蛋白质-DNA 结合能量之间的联系提供第一个直接的结构和量热测量。这是一个基础研究项目，将描述许多疾病相关的蛋白质-DNA 复合物中发生的一种重要 DNA 相互作用的能量学。 Sac7d 是一个染色结构域，是真核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。该蛋白质通过与具有直接生物医学相关性的蛋白质中观察到的机制类似的机制与 DNA 结合。这些结果将增强我们合理控制相关蛋白质-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目标。