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）：Kelly Lee 建议确定 蛋白质亚基大规模、pH 依赖性重组的机制 RNA病毒，NomegaV。我们实验室之前的工作展示了非凡的 病毒样颗粒的尺寸和四级结构变化 当 pH 从 7 降至 5 时，杆状病毒表达系统。Kelly 将 利用他在高分辨率电位滴定方面的经验来确定 转变过程中被质子化的酸性基团的数量。最近一个 制备的突变体允许转变是可逆的，这将被研究 滞后迹象。基于这些和低的可用结构 凯利将提出病毒的 pH 形式的关键酸性残留物的候选者 在过渡中。实验室的研究生德里克·泰勒 (Derek Taylor) 将制作 这些残基发生突变，凯利将研究它们的表型。凯莉将会 通过表征假定的中间体进行结构方法培训 通过冷冻电镜和图像重建进行转变，这些将通过 以低 pH 形式确定的亚基的高分辨率 X 射线结构。 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 端 73 个氨基酸部分进行定点突变 子单位，被怀疑在转变中很重要。该多肽 还将在溶液中作为与 GST 的融合蛋白进行研究，看看它是否具有 pH 依赖性寡聚行为。凯利假设它是三聚体 高pH值时为单体，低pH值时为单体。我们预计我们将能够 通过选定的突变控制转变，我们将绘制 当凯利完成他的研究时蛋白质重组的轨迹 奖学金。