Assembly and Structure of Bacteriophage Lambda

描述(申请人提供)：这项资助的主要重点是了解蛋白质纳米机器是如何由其部件组装而成的，组装后如何发挥作用，以及组装机制如何支持纳米机器的生物功能。为了解决这些问题，我们将使用噬菌体HK97的衣壳和噬菌体的尾巴？这两者都提供了生物组装的核心重要方面的实验途径：HK97衣壳蛋白的Delta结构域是一个组装伴侣，它介导衣壳组装成衣壳，并结合蛋白酶并将其带入衣壳内部。它还在使门户就位方面发挥作用，并可能因此在形成衣壳组装的核中起到关键作用。对于尾巴，我们最近发现的尾巴组装伴侣以及它们与卷尺蛋白和尾管蛋白相互作用的方式，以一种以前不清楚的方式阐明了尾巴组装的主要特征。我们的两个模型系统也提供了如何在生物组装中确定大小的例子。HK97组装T=7衣壳蛋白，但具有非常相似衣壳蛋白序列的D3组装T=9。尾巴组装到我们多年前展示的长度由卷尺蛋白决定。我们最近对组装伴侣的研究结果阐明了卷尺测量蛋白质中的长度信息是如何转化为尾巴长度的。我们建议对HK97头进行的实验将包括产生衣壳蛋白的突变体，以改变衣壳成熟的过程。将根据它们的生化和结构表型进行评估，并在早期研究提供的丰富的结构和结构/动态数据的背景下进行解释。我们将特别关注衣壳蛋白的Delta结构域，它在衣壳蛋白功能的几个方面发挥着关键作用。对于尾巴，我们最初将专注于研究新发现的组装中间体，即卷尺测量蛋白和两个组装伴侣的复合体，我们将检查其结构和组装活性。我们将开始实验来确定尾端的结构和功能，包括次要蛋白和新发现的铁-硫簇。我们建议表征两个非规范翻译的头部蛋白(TerL)的例子，并对新发现的一种未知类型的噬菌体进行基本表征。

DESCRIPTION (provided by applicant): The main emphasis of this grant is to understand how a protein nanomachine is assembled from its parts, how it functions once assembled, and how the mechanisms of assembly support the nanomachine's biological functions. To address these questions we will use the capsid of bacteriophage HK97 and the tail of bacteriophage??? Both of these offer experimental access to centrally important aspects of biological assembly: The Delta domain of the HK97 capsid protein is an assembly chaperone that mediates assembly of capsomers into capsid and also binds the protease and brings it into the inside of the capsid. It also has a role in getting the portal into position and probably has a crucial role in nucleating capsid assembly as a result. For the tails our recent discovery of the tail assembly chaperones and the way they interact with the tape measure protein and with the tail tube protein illuminates the main features of tail assembly in a way that was not previously clear. Both of our model systems also provide examples of how size can be determined in biological assembly. HK97 assembles T=7 capsids but D3, which has a very similar capsid protein sequence, assembles a T=9. The tail assembles to a length that we showed years ago is determined by the tape measure protein. Our recent result with the assembly chaperones clarifies how the length information in the tape measure protein is translated into tail length. Our proposed experiments with the HK97 heads will include generating mutants of the capsid protein chosen to alter the process of capsid maturation. These will be evaluated by their biochemical and structural phenotypes and interpreted in the context of the wealth of structural and structural/dynamic data available from earlier studies. We will pay particular attention to the Delta domain of the capsid protein, which has crucial roles in several aspects of capsid protein function. For tails we will concentrate initially on investigating the newly identified assembly intermediate, a complex of the tape measure protein and the two assembly chaperones, for which we will examine the structure and assembly activities. We will begin experiments to determine the structure and function of the tail tip, including the minor proteins and the newly discovered iron-sulfur cluster We propose to characterize two examples of non-canonical translation of a head protein (TerL), and to carry out a basic characterization of a newly discovered bacteriophage of a previously unknown type.