Assembly of Single Biological Molecular Machines

描述(申请人提供)：各种生物分子机器的组装和功能是细胞结构和功能的关键。由于它们的复杂性，这些机器组装的机械细节往往被隐藏在总体实验中。在这个项目中，我们将开发和使用多色单分子FRET方法来研究一个重要的分子机器，细菌核糖体的30S亚单位。三色和四色FRET方法将提供构象变化和结合的耦合信息，以及组装亚基中分离良好的部分的构象变化。我们将使用这些单分子方法，通过研究一系列不同大小和复杂性的核糖体结构，在多个水平上探索组装过程。我们将首先从平台域探索小蛋白结合模块的构象变化。我们还将研究分离的头部和整个亚基作为蛋白质结合的函数的全局变化，以及组装路径上的关键中间体。我们希望这些研究将提供新的方法和对组装的见解，这将对普通生物界有用，也可能有助于理解如何设计改进的抗生素。

DESCRIPTION (provided by applicant): The assembly and function of a variety of biological molecular machines is key to the structure and function of the cell. Because of their complexity, mechanistic details of the assembly of these machines is often hidden in ensemble experiments. In this project, we will develop and use multicolor single molecule FRET methods to study an important molecular machine, the 30S subunit of the bacterial ribosome. 3- and 4-color FRET methods will provide information about coupling of conformational changes and binding, as well as conformational changes in well-separated parts of the assembling subunit. We will use these single-molecule methods to probe the assembly process at multiple levels, by studying a series of ribosomal constructs of different size and complexity. We will first probe conformational changes in small protein-binding modules from the platform domain. We will also study global changes in the isolated head and the entire subunit as a function of protein binding as well as at key intermediates along the assembly pathway. We expect that these studies will provide new methods and insights into the assembly that will be useful to the general biological community, and also might help understand how to design improved antibiotics.