Mechanistic studies of protein reorganization in a virus

病毒中蛋白质重组的机制研究

• 批准号: 6622380
• 负责人: KELLY K LEE
• 金额: $ 4.16万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6622380
• 关键词: Baculoviridae; RNA virus; X ray crystallography; acidity /alkalinity; analytical method; capsid; conformation; cryoelectron microscopy; host organism interaction; intermolecular interaction; model design /development; molecular assembly /self assembly; molecular shape; physical model; postdoctoral investigator; protein structure function; protonation; site directed mutagenesis; structural biology; virus genetics; virus infection mechanism; virus morphology; virus protein; viruslike particle

DESCRIPTION (provided by applicant): Kelly Lee proposes to determine the mechanism of large scale, pH dependent reorganization of protein subunits in an RNA virus, NomegaV. Previous work in our laboratory demonstrated a remarkable size and quaternary structure change of virus-like particles made in a baculovirus expression system when the pH was lowered from 7 to 5. Kelly will use his experience in high resolution potentiometric titrations to determine the number of acidic groups being protonated during the transition. A recently prepared mutant allows the transition to be reversible and this will be studied for signs of hysteresis. Based on these and the available structure of the low pH form of the virus Kelly will propose candidates for critical acidic residues in the transition. Derek Taylor, a graduate student in the lab will make mutations to these residues and Kelly will study their phenotype. Kelly will be trained in structural methods by characterizing putative intermediates in the transition by cryoEM and image reconstruction and these will be modeled by the high resolution x-ray structure of the subunit determined in the low pH form. Kelly will also learn methods of molecular biology and baculovirus expression by making site directed mutations in the C-terminal 73 amino acid portion of the subunit, suspected of being important in the transition. This polypeptide will also be studied in solution as a fusion protein with GST to see if it has a pH dependent oligomeric behavior. Kelly has hypothesized that it is a trimer at high pH and a monomer at low pH. We anticipate that we will be able to control the transition by selected mutations and that we will map the trajectory of protein reorganization by the time Kelly has completed his fellowship.

描述（由申请人提供）：凯利·李（Kelly Lee）提议确定 大规模的机制，pH依赖性的蛋白质亚基的重组 RNA病毒，Nomegav。我们实验室的先前工作表现出了非凡的 在A 当pH从7降低到5时，杆状病毒表达系统。 利用他在高分辨率电位测量滴定方面的经验来确定 在过渡过程中质子化的酸性基团的数量。最近 准备好的突变体允许过渡是可逆的，这将被研究 用于磁滞的迹象。基于这些以及低的可用结构 病毒Kelly病毒的pH形式将提出关键酸性残基的候选者 在过渡中。实验室的研究生德里克·泰勒（Derek Taylor）将成为 这些残基的突变和凯利将研究其表型。凯利会 通过表征在结构方法中训练的结构方法 冷冻和图像重建的过渡，这些将由 亚基的高分辨率X射线结构以低pH形式确定。 凯利还将学习分子生物学和杆状病毒表达的方法 通过在C末端73氨基酸部分中进行位置定向突变 涉嫌在过渡中很重要的亚基。这个多肽 还将在溶液中作为与GST的融合蛋白进行研究，以查看其是否具有 pH依赖性的寡聚行为。凯利（Kelly）假设这是一个三聚机 在高pH值和低pH值的单体处。我们预计我们将能够 控制选定突变的过渡，我们将绘制 凯利完成了蛋白质重组的轨迹 奖学金。