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: 7430686
• 负责人: Andreas Hanke
• 金额: $ 10.13万
• 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/7430686
• 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; Condition; 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; Helix (Snails); Investigation; Kinetics; Light; Localized; Measurement; Measures; Methods; Modeling; Molecular Biology; Nucleotides; Numbers; Pharmaceutical Preparations; Pilot Projects; Process; Property; Protein Binding; Proteins; Publications; Range; Rate; Regulation; Research; Role; SS DNA BP; Score; Single-Stranded DNA; Solvents; Son of Sevenless Proteins; Spectrum Analysis; Stretching; Structure; Techniques; Temperature; Thermodynamics; Time; Traction; Universities; Work; design; ds-DNA; ear helix; infancy; instrument; laser tweezer; melting; mutant; novel; research study; single molecule; size; 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（由申请人提供）：DNA的结构转换和蛋白质在DNA上的对接是分子生物学中的基本过程。理解和控制这些过程对于设计抗感染性疾病的新药至关重要（例如，HIV）。为了更好地理解DNA复制的生物学过程，重要的是研究单个DNA分子和在细胞中发生的不同条件下与DNA相互作用的分子的性质。单分子力谱已被证明是一种多功能的技术，用于调查DNA二级结构的变化，然而，这种技术的应用仍处于起步阶段。目前，单双链体DNA分子通过光学镊子仪器拉伸，其在约65 pN的拉伸力下可以诱导DNA双链体的两条链的解旋（螺旋-卷曲转变）。然后可以在广泛的条件下探测由牵引产生的解绕DNA的局部区域与单链DNA结合蛋白的相互作用。这些新的实验技术的补充，最近发展的先进的生物物理和计算模型的螺旋线圈过渡。具体而言，噬菌体T7基因2.5蛋白（gp2.5）是一种单链DNA结合蛋白，在噬菌体感染的细胞中的DNA复制和重组中具有重要作用。通过研究在gp2.5存在下的速率依赖性DNA解链力，在蛋白质缔合和解离速率以及平衡缔合常数方面测量蛋白质与单链DNA结合的动力学和热力学。该测量的一个重要参数是作为测量时间的函数的DNA中的螺旋-卷曲边界在过度拉伸转变处的数目。 本研究的主要目的是建立一个全面的生物物理模型，拉伸DNA及其与单链DNA结合蛋白的相互作用。具体来说，我们将使用这个模型来确定上述实验参数。该项目的结果有望为传染病的基本生物过程提供新的线索。该项目是S 06 SCORE试点项目的延续，具有相同的总体范围。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。