CAREER: Target-Site Search of DNA-Binding Proteins

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。在这篇CAREER proposal中，PI提议研究dna结合蛋白的靶点搜索策略。PI将开发一种反应扩散方法来模拟蛋白质找到其靶点的促进扩散。反应-扩散理论将捕获蛋白质动力学和DNA的构象行为，建立一个蛋白质易位的预测，可以适应不同的DNA物理模型。该理论将通过结合促进扩散锁定第一个结合位点和随后的环化机制结合第二个结合位点，扩展到2位点结合蛋白。PI提出了在2位点结合蛋白中形成复合物的两种状态机制，包括热诱导环和由于DNA与第二个蛋白质结合位点的非特异性结合而产生的捆绑机制。超螺旋DNA的分析理论和布朗动力学模拟将用于建立一个理论框架来预测质粒DNA中超螺旋的成核和生长，这将用于解决1位点和2位点结合蛋白结合动力学中血小板素形成和重组的影响。与理论工作相结合，将开发一个综合实验计划来测试理论预测并指导模型开发。利用限制性内切酶作为靶位定位的探针，将进行实验来验证理论预测和提出的靶位搜索机制。这项研究计划代表了一种多方面的方法来理解蛋白质如何利用非特异性结合来增强动力学，这是小数量生物学的核心。外联工作包括为正在接受儿童癌症治疗的高中生制定和实施教学模块。在暑假期间，PI还将与一名高中教师合作，以获得关于实施的难易程度和学生对教学模块理解程度的反馈。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).In this CAREER proposal the PI, proposes to study the target-site search strategy of DNA-binding proteins. The PI will develop a reaction-diffusion methodology to model the facilitated diffusion of a protein finding its target site. The reaction-diffusion theory will capture both the protein dynamics and the conformational behavior of the DNA, building a prediction for protein translocation that can be adapted to different physical models of DNA. The theory will be extended to 2-site binding proteins by combining facilitated diffusion to lock in the first binding site and a subsequent looping mechanism to bind the second site. The PI proposes a two-state mechanism for complex formation in 2-site binding proteins that includes thermal induced looping and a cinching mechanism due to non-specific binding of DNA to the second protein binding site. Analytical theory and Brownian dynamics simulations of supercoiled DNA will be used to develop a theoretical framework to predict the nucleation and growth of supercoils in plasmid DNA, which will be used to address the impact of plectoneme formation and restructuring in the binding kinetics of 1-site and 2-site binding proteins. In conjunction with the theoretical efforts, an integrated experimental program to test the theoretical predictions and guide model development will be developed. Using restriction enzyme digestions as a probe for target-site localization, experiments will be performed to verify the theoretical predictions and the proposed mechanisms for the target-site search. This research proposal represents a multi-faceted approach to understanding how proteins exploit non-specific binding to enhance kinetics, which is central in the biology of small numbers. Outreach efforts include the development and implementation of teaching modules into the education of high school students that are being treated for childhood cancer. The PI will also pair with a high school teacher during the summers to receive feedback on the ease of implementation and student understanding of the teaching modules.