Protein Diffusion and Interaction in Concentrated DNA Solutions

9728165布卢姆菲尔德脱氧核糖核酸在拥挤的、强相互作用的溶液中发挥作用，主要是在与蛋白质相互作用时。蛋白质-DNA相互作用的平衡和动态方面受到环境的强烈影响。当溶液的体积占有率达到30%以上时，就像它在细胞内所做的那样，根据稀溶液行为的简单外推变得完全不够用。这个项目的目标是更好地从实验和理论上理解高浓度的DNA和其他聚合物如何影响与DNA结合的蛋白质的扩散动力学。这三个具体目标是(1)表征蛋白质在拥挤的DNA环境中以及在也含有聚集剂如葡聚糖、菲科尔和聚乙二醇酯的DNA溶液中的扩散运动，以了解排除体积、离子相互作用和特定结合相互作用的影响。(2)确定拥挤如何影响限制酶作用中一维和三维扩散的相对重要性。(3)用布朗动力学模拟蛋白质在拥挤的DNA溶液中的扩散，了解离子强度依赖的体积排斥和非特异性结合如何影响调节蛋白质寻找其特定结合部位。主要的工具将是荧光光漂白恢复和动态光散射来测量扩散，凝胶电泳法来跟踪限制酶作用的动力学，以及布朗动力学来模拟复杂、相互作用的体系中的扩散。这项研究的总体意义是深入了解细胞内普遍存在的拥挤、强烈相互作用的条件的影响，以及这些条件如何影响涉及基因调控的动态扩散和反应过程。***

9728165 Bloomfield DNA functions in crowded, strongly interacting solutions, mainly while interacting with proteins. The equilibrium and dynamic aspects of protein-DNA interactions are strongly influenced by the environment. When the volume occupancy of a solution reaches more than thirty percent, as it does within cells, simple extrapolations from dilute solution behavior become totally inadequate. The goal in this project is to develop a better experimental and theoretical understanding of the ways in which high concentrations of DNA and other polymers influence the diffusional dynamics of proteins which bind to DNA. The three specific aim are (1) To characterize the diffusional motion of proteins within a crowded DNA environment and in DNA solutions which also contain crowding agents such as dextran, Ficoll, and PEG, so as to understand the effects of excluded volume, ionic interactions, and specific binding interactions. (2) To determine how crowding influences the relative importance of one-dimensional and three-dimensional diffusion in the action of restriction enzymes. (3) To use Brownian dynamics simulation of protein diffusion in crowded DNA solutions, to gain insight into the ways in which ionic strength-dependent volume exclusion and nonspecific binding affect the search of regulatory proteins for their specific binding sites. The major tools will be fluorescence photobleaching recovery and dynamic light scattering to measure diffusion, gel electrophoresis to follow kinetics of restriction enzyme action, and Brownian dynamics to simulate diffusion in complex, interacting systems. The general significance of this research is to gain insight into the effect of the crowded, strongly interacting conditions prevailing inside cells, and how these conditions affect the dynamic diffusion and reaction processes involved in gene regulation. ***