Energetics of Protein-DNA Binding and Bending

描述(申请人提供)：蛋白质诱导的DNA扭曲对许多正常的细胞功能是必不可少的，包括基因调节、表达、重组和染色质组织方面。DNA扭曲是一个耗费能量的过程，与蛋白质-DNA复合体的亲和力和功能密切相关。对蛋白质引起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，这是因为在既可以获得量热数据又可以获得结构数据的络合物中，很难控制DNA扭曲和弯曲作为实验变量。超热稳定的Sac7d-DNA复合体非常适合作为定量研究由于微小凹槽结合而导致的DNA弯曲和解卷的能量学的模型基准系统。在这个提议中，我们关注的是这样一个事实，即在Sac7d-DNA复合体中可以实验地操纵DNA扭曲的水平，其中可以跟踪结构变化和能量学。我们的目标是定义Sac7d在溶液中诱导的DNA扭曲，并将扭曲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的扭曲和结合能的影响。此外，我们将研究细胞条件，包括盐浓度、特定的反离子和渗透压，对扭曲的大小和相关的结合和弯曲的能量的影响。这将为DNA扭曲与蛋白质-DNA结合的能量学之间的联系提供第一个直接的结构和量热方法。这是一个基础研究项目，将描述在许多与疾病相关的蛋白质-DNA复合体中发生的一种重要类型的DNA相互作用的能量学。Sac7d是一个染色域，在真核细胞核蛋白中普遍存在，也存在于HIV-1整合酶的DNA结合域中。这种蛋白质与DNA的结合机制类似于在蛋白质中观察到的与直接生物医学相关的机制。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.