PROTEIN POLYMORPHISM DYNAMICS IN ICOSAHEDRAL VIRUSES

二十面体病毒中的蛋白质多态性动力学

• 批准号: 6386318
• 负责人: John Emil Johnson
• 金额: $ 33.43万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386318
• 关键词: Bromovirus; RNA virus; X ray crystallography; biological polymorphism; biophysics; capsid; computer simulation; conformation; cryoelectron microscopy; crystallization; insect virus; mass spectrometry; molecular dynamics; mutant; physical model; protein protein interaction; protein structure; structural biology; time resolved data; virion; virus RNA; virus assembly; virus morphology; virus protein; viruslike particle

We propose to continue our studies of protein polymorphism in virus particles and to extend these to the dynamic analysis of quaternary structure transitions amenable to investigation by time-resolved methods. Our goal is to characterize the tertiary structures of the subunits and to identify the molecular switches and chemical surface features that make them suited for multiple quaternary structure conformations. In each case we have or are determining the near-atomic resolution structure of at least one form of the particle by x-ray crystallography. If alternate polymorphs can not be crystallized because they are moderately heterogeneous or unstable, the atomic subunit model derived from the high resolution structure will be used to develop pseudo atomic resolution structures of the polymorphs with methods described below. All of the systems that we are investigating can be assembled either in vitro or as a recombinant assembly (virus-like-particles; VLPs) within the cells of the heterologus expression system used. Thus our hypotheses from detailed modeling and physical studies can be directly tested by molecular genetic alterations and the analysis of the consequent assembly phenotype. The study of static polymorphs of viral subunits from alfalfa mosaic virus (AMV), cowpea chlorotic mottle virus (CCMV), rice yellow mottle virus (RYMV), flock house virus (FHV), Nudaurelia capensis omega virus (NomegaV) and LA virus will be initiated or extended by a variety of methods. These methods include (a) conventional or ultra-low resolution x-ray crystallography, (b) cryo-electron microscopy and image reconstruction, (c) molecular modeling with high resolution coordinates into low resolution experimental density functions using texture surface mapping and a generalized symmetry server, (d) refinement of derived models by molecular mechanics and energy minimization, (e) solution x-ray scattering and data analysis with icosahedral or spherical harmonics, and (f) proteolytic susceptibility studies with resulting polypeptides analyzed by mass spectrometry. Systems amenable to time resolved analysis include the reversible swelling of CCMV and RYMV and the large scale quaternary structure transition in NomegaV. These will be investigated by time resolved solution x-ray scattering (100 millisecond regime) and cryoEM (500millisecond regime). The long-term goal of these studies is to find the means to interfere with these transitions through the use of rationally designed small molecules. We have chosen the model systems described for their experimental accessibility, but anticipate that their study, in the context described, will provide a general and basic understanding of the principles of protein polymorphism and the ability to specifically alter such transitions.

我们建议继续对病毒颗粒中蛋白质多态性的研究，并将其扩展到适合时间分辨方法研究的四级结构转变的动态分析。 我们的目标是表征亚基的三级结构，并确定使它们适合多种四级结构构象的分子开关和化学表面特征。 在每种情况下，我们已经或正在通过 X 射线晶体学确定至少一种形式的粒子的近原子分辨率结构。 如果替代多晶型物由于中等异质性或不稳定而不能结晶，则将从高分辨率结构导出的原子亚基模型将用于通过下述方法开发多晶型物的伪原子分辨率结构。 我们正在研究的所有系统都可以在体外进行组装，也可以在所使用的异源表达系统的细胞内进行重组组装（病毒样颗粒；VLP）。 因此，我们通过详细建模和物理研究提出的假设可以通过分子遗传改变和随后的组装表型分析来直接测试。苜蓿花叶病毒（AMV）、豇豆褪绿斑驳病毒（CCMV）、水稻黄斑驳病毒（RYMV）、鸡舍病毒（FHV）、Nudaurelia capensis omega病毒（NomegaV）和LA病毒的病毒亚基静态多态性的研究将通过多种方法启动或扩展。 这些方法包括（a）传统或超低分辨率X射线晶体学，（b）冷冻电子显微镜和图像重建，（c）使用纹理表面映射和广义对称服务器将高分辨率坐标分子建模为低分辨率实验密度函数，（d）通过分子力学和能量最小化对导出模型进行细化，（e）溶液X射线散射和数据分析 二十面体或球谐函数，以及（f）通过质谱分析所得多肽的蛋白水解敏感性研究。 适合时间分辨分析的系统包括 CCMV 和 RYMV 的可逆膨胀以及 NomegaV 中的大规模四级结构转变。 这些将通过时间分辨溶液 X 射线散射（100 毫秒范围）和冷冻电镜（500 毫秒范围）进行研究。 这些研究的长期目标是找到通过使用合理设计的小分子来干扰这些转变的方法。 我们选择了所描述的模型系统是为了其实验可访问性，但预计他们的研究在所描述的背景下将提供对蛋白质多态性原理的一般和基本理解以及特异性改变此类转变的能力。