Structure & Function of Bacteriophage Portal Proteins

智力价值该项目专注于破译病毒衣壳组装过程中发生的蛋白质/蛋白质相互作用的序列，使用噬菌体phi29作为模型系统。病毒衣壳是由数百个化学上相同的蛋白质分子自组装而成的保护性蛋白质外壳。在最终的衣壳中，这些分子精确地定位在空间中，呈整体球形。蛋白质壳包围并保护病毒核酸，在一种衣壳中，壳首先组装，然后核酸通过一种称为入口的管道被泵入壳中。尽管这种结构主题无处不在，但人们对蛋白质自组装的蛋白质/蛋白质相互作用的途径或序列知之甚少。最近的实验数据表明，组装来自两个蛋白质的多个副本组成的复合体，一个支架蛋白和形成门户蛋白的管道。该项目将使用化学交联/质谱学、氢/氚交换研究和突变分析，并结合计算对接来确定这种成核复合体的详细结构，并利用这些信息在化学上稳定成核复合体。然后，稳定的成核复合体将被用于在整体溶液和单分子实验中播撒组装反应，以探索亚基与生长衣壳添加的序列和动力学，并获得对组装途径的分子水平的理解。广泛的重要的是，正在开发以病毒为范例的自组装模型的物理学家和数学家干部需要对病毒衣壳组装的分子途径进行详细的定量描述，而目前还没有对任何病毒提供这种详细程度的描述。该研究项目本身使用了各种各样的生物物理和生化工具，是将进行实验的研究生和本科生的理想培训平台。最后，由于详细的分子路径是最好的欣赏和理解，当动画说明时，本项目定义的组装路径将由艺术系和艺术史系的学生为外行观众制作动画，让他们接触一线科学，同时允许他们完善他们的动画技能和开发一个投资组合。

Intellectual MeritThe project focuses on deciphering the sequence of protein/protein interactions which occur during the assembly of viral capsids using the bacteriophage phi29 as a model system. Viral capsids are protective protein shells that self assemble from hundreds of chemically identical protein molecules. In the final capsid these molecules are precisely positioned in space with an overall spherical form. The protein shell surrounds and protects the viral nucleic acid and in one class of capsid the shell assembles first and the nucleic acid is subsequently pumped into the shell through a conduit known as a portal. Despite the ubiquity of this architectural theme, little is known about the pathway or sequence of protein/protein interaction through which the proteins self-assemble. Recent experimental data suggests that assembly nucleates from a complex composed of multiple copies of two proteins, a scaffolding protein and the conduit forming portal protein. The project will use chemical cross-linking/mass spectrometry, hydrogen/deuterium exchange studies and mutational analysis in concert with computational docking to determine the detailed structure of this nucleation complex, and use this information to chemically stabilize the nucleation complex. The stabilized nucleation complexes will then be used to seed assembly reactions both in bulk solution and in single molecule experiments to probe the sequence and kinetics of subunit addition to growing capsids and derive a molecular level understanding of the assembly pathway.Broader ImpactA detailed quantitative description of the molecular pathway of viral capsid assembly is required by the cadre of physicist and mathematicians who are developing models of self-assembly using viruses as a paradigm and a description at this level of detail is not currently available for any virus. The research project itself employs a wide variety of biophysical and biochemical tools and serves as an ideal training platform for the graduate and undergraduate students who will be carrying out the experiments. Finally, because detailed molecular pathways are best appreciated and understood when illustrated by animation the assembly pathway defined by this project will be animated for a lay audience by students in the Department of Art and the Art History Department exposing them to frontline science while allowing them to refine their animation skills and develop a portfolio.