Structure and Function of Bacteriophage Proteins

噬菌体蛋白的结构和功能

• 批准号: 1515260
• 负责人: Richard Kuhn
• 金额: $ 158.41万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1515260&HistoricalAwards=false
• 关键词: Structure; Function; Bacteriophage; Proteins

A virus that infects bacteria (known as a bacteriophage) must be able to recognize and attach to the specific host cell and then trigger a mechanism by which the viral genome (its nucleic acid) is injected into the host cell. The viral nucleic acid is packaged inside a protein coat called the capsid, and the majority of bacteriophages are assembled with a tail on the capsid through which the viral nucleic acid can be injected into the host cell to infect it. It has recently become apparent that members of the Microviridae (a family of small single stranded DNA bacteriophages) generate their tail at the time of infection rather than at the time of initial phage assembly. This project is directed at investigating this process and comparing it with the structural and functional properties of bacteriophage C1, another type of bacteriophage that has a fixed tail. The analysis of bacteriophages provides insight into the structure and function of extraordinarily intricate molecular machines, which could provide insight for innovations in biotechnology. This project will provide interdisciplinary training to students and postdoctoral fellows at the interface of biophysics and biology. The structure and function of the small, icosahedral phiX174 bacteriophage is to be compared with that of the podovirus C1, using X-ray crystallography to study the viral components and cryo-electron microscopy as well as electron tomography to study the viruses as they invade their host bacteria. This project has two specific aims. Specific Aim 1: The mechanism by which the phiX174 genome translocates its genome through the phage encoded H tube into an E. coli host cell is to be determined. a. The state of assembly of the tail tube in phiX174 virions is to be determined. The assembled tail consists of ten copies of the virally encoded H protein that form a coiled coil structure. b. The interaction between phage ST-1 (a close relative of phiX174) and host cell lipopolysaccharide molecules is to be determined using cryoEM and cryoTM. ST-1 needs to be used for these experiments because the E. coli hosts for phiX174 are too thick to allow the electron beam to penetrate without too much attenuation. c. The H-tube assembly is to be studied, by changing the relative lengths of the A and B domains and by modifying the lengths of the H-tubes. d. The structures of the H protein in other, distantly related Microviridae are to be determined. e. The systematically occurring Gln residues in the DNA conduit of phiX174 and phage T7 are to be changed in order to establish their function. f. Complexes between the H tubes and ssDNA are to be analyzed. Specific Aim 2: The structure of bacteriophage C1 is to be determined. a. A near-atomic resolution structure of the C1 head is to be determined using cryoEM. b. The structure of the C1 tail proteins are to be determined using X-ray crystallography.

感染细菌的病毒（称为噬菌体）必须能够识别并附着在特定的宿主细胞上，然后触发一种机制，通过这种机制，病毒基因组（其核酸）被注射到宿主细胞中。病毒核酸被包装在一种叫做衣壳的蛋白质外壳内，大多数噬菌体在衣壳上装配有一条尾巴，病毒核酸可以通过这条尾巴注射到宿主细胞中去感染它。最近发现，微病毒科（一个小单链DNA噬菌体家族）的成员在感染时而不是在初始噬菌体组装时产生它们的尾巴。本项目旨在研究这一过程，并将其与另一种具有固定尾巴的噬菌体C1的结构和功能特性进行比较。对噬菌体的分析提供了对极其复杂的分子机器的结构和功能的深入了解，这可能为生物技术的创新提供见解。本项目将为生物物理学和生物学交叉领域的学生和博士后提供跨学科的培训。将小的二十面体phiX174噬菌体与足病毒C1噬菌体的结构和功能进行比较，使用x射线晶体学研究病毒成分，使用低温电子显微镜和电子断层扫描研究病毒入侵宿主细菌的过程。这个项目有两个具体目标。特异性目的1:phiX174基因组通过噬菌体编码的H管将其基因组易位到大肠杆菌宿主细胞的机制有待确定。a.待确定phiX174病毒粒子中尾管的装配状态。组装的尾部由10个病毒编码的H蛋白副本组成，形成一个盘绕的螺旋结构。b.噬菌体ST-1 （phiX174的近亲）与宿主细胞脂多糖分子的相互作用将使用cryoEM和cryoTM进行测定。ST-1需要用于这些实验，因为phiX174的大肠杆菌宿主太厚，无法让电子束穿透而不产生太多衰减。c.通过改变A和B结构域的相对长度以及修改h管的长度来研究h管组装。d.其他亲缘关系较远的微病毒科中H蛋白的结构有待确定。e.改变phiX174和噬菌体T7 DNA导管中系统存在的Gln残基，以建立其功能。f.要分析H管和ssDNA之间的配合物。具体目的2：确定噬菌体C1的结构。a.使用cryoEM确定C1头的近原子分辨率结构。b.使用x射线晶体学确定C1尾部蛋白的结构。