Thermodynamics and kinetics of DNA binding protein probed by DNA overstretching

DNA 过度拉伸探测 DNA 结合蛋白的热力学和动力学

• 批准号: 7618819
• 负责人: Andreas Hanke
• 金额: $ 10.18万
• 依托单位: UNIV/TEXAS BROWNSVILLE & SOUTHMOST COLL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7618819
• 关键词: Advanced Development; Bacteriophage T7; Bacteriophages; Base Pairing; Binding; Binding Sites; Biological Process; Boston; C-terminal; Cells; Collaborations; Communicable Diseases; Complement; Computer Simulation; DNA; DNA Probes; DNA biosynthesis; DNA-Binding Proteins; Data; Dependence; Development; Diffusion; Dissociation; Docking; Enterobacteria phage T7 gene 2.5 protein; Equilibrium; Faculty; Free Energy; Future; Gene Proteins; Genes; Genetic Recombination; HIV; Investigation; Kinetics; Light; Measurement; Measures; Methods; Modeling; Molecular Biology; Nucleotides; Pharmaceutical Preparations; Pilot Projects; Process; Property; Protein Binding; Proteins; Publications; Regulation; Research; Role; SS DNA BP; Single-Stranded DNA; Solvents; Son of Sevenless Proteins; Spectrum Analysis; Stretching; Structure; Techniques; Temperature; Thermodynamics; Time; Traction; Universities; Work; design; ds-DNA; infancy; instrument; laser tweezer; melting; mutant; novel; research study; single molecule; tool

DESCRIPTION (provided by applicant): Structural transitions of DNA and docking of proteins on DNA are fundamental processes in molecular biology. Understanding and controlling these processes are essential to the design of novel drugs against infectious diseases (e.g., HIV). In order to better understand the biological process of DNA replication, it is important to study the properties of single DNA molecules and molecules that interact with DNA under different conditions occurring in the cell. Single-molecule force spectroscopy has proved to be a versatile technique for investigating changes in DNA secondary structure; however, applications of this technique are still in their infancy. Currently, single duplex DNA molecules are stretched by an optical tweezers instrument, which at a stretching force of about 65 pN can induce the unwinding of the two strands of the DNA duplex (helix-coil transition). The interaction of the localized regions of unwound DNA generated by traction with single-stranded DNA binding proteins can then be probed under a wide range of conditions. These new experimental techniques are complemented by the recent development of advanced biophysical and computational models for the helix-coil transition. Specifically, Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA binding protein that has essential roles in DNA replication and recombination in phage-infected cells. By studying the rate-dependent DNA melting force in the presence of gp2.5, the kinetics and thermodynamics of protein binding to single-stranded DNA is measured in terms of protein association and dissociation rates and the equilibrium association constant. An important parameter for this measurement is the number of helix-coil boundaries in the DNA at the overstretching transition as a function of measurement time. The main objective of this research is to develop a comprehensive biophysical model for stretched DNA and its interaction with single-stranded DNA binding proteins. Specifically, we will use this model to determine the experimental parameter mentioned above. The results of this project are expected to shed new light on fundamental biological processes of infectious diseases. This project is the continuation of a S06 SCORE pilot project with the same general scope. It is a starting point for future long-term collaborative research and will therefore be an important tool for the PI's faculty development.

描述(申请人提供)：DNA的结构转变和蛋白质在DNA上的对接是分子生物学中的基本过程。了解和控制这些过程对于设计治疗传染病(如艾滋病毒)的新药至关重要。为了更好地理解DNA复制的生物学过程，研究单个DNA分子和在细胞内不同条件下与DNA相互作用的分子的性质是很重要的。单分子力谱已被证明是研究DNA二级结构变化的一种通用技术；然而，该技术的应用仍处于起步阶段。目前，单链双链DNA分子是通过光镊仪拉伸的，在大约65pN的拉伸力下，可以诱导DNA双链的两条链解开(螺旋-线圈转变)。然后，可以在广泛的条件下探索由牵引产生的未缠绕DNA的局部区域与单链DNA结合蛋白的相互作用。这些新的实验技术得到了螺旋-线圈转变的先进生物物理和计算模型的最新发展的补充。特别是，噬菌体T7基因2.5蛋白(gp2.5)是一种单链DNA结合蛋白，在噬菌体感染细胞中对DNA复制和重组具有重要作用。通过研究gp2.5存在下的速度依赖性DNA熔融力，测量了蛋白质与单链DNA结合的动力学和热力学，包括蛋白质的缔合和解离速率以及平衡缔合常数。这项测量的一个重要参数是DNA在过度拉伸转变时作为测量时间函数的螺旋-螺旋边界的数量。 本研究的主要目的是为拉伸DNA及其与单链DNA结合蛋白的相互作用建立一个全面的生物物理模型。具体来说，我们将利用这个模型来确定上面提到的实验参数。该项目的结果有望为传染病的基本生物学过程提供新的线索。这个项目是S06成绩试点项目的延续，具有相同的一般范围。它是未来长期合作研究的起点，因此将成为PI教师发展的重要工具。