PROTEIN POLYMORPHISM DYNAMICS IN ICOSAHEDRAL VIRUSES

我们建议继续我们的研究病毒颗粒中的蛋白质多态性，并将其扩展到适合于时间分辨方法调查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，并确定分子开关和化学表面特征，使它们适合于多个四级结构构象。 在每一种情况下，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。 如果替代的多晶型物因为它们是中等不均匀的或不稳定的而不能结晶，则从高分辨率结构衍生的原子亚基模型将用于用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组装或在所使用的异源表达系统的细胞内作为重组组装体（病毒样颗粒; VLP）组装。 因此，我们的假设，从详细的建模和物理研究可以直接测试的分子遗传改变和随之而来的组装表型的分析。苜蓿花叶病毒（AMV）、豇豆褪绿斑驳病毒（CCMV）、水稻黄斑驳病毒（RYMV）、鸡场病毒（FHV）、角裸尾壳病毒（NomegaV）和LA病毒的亚单位的静态多态性研究将通过多种方法启动或扩展。 这些方法包括（a）常规或超低分辨率X射线晶体学，（B）低温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的分子建模成低分辨率实验密度函数，（d）通过分子力学和能量最小化来细化导出的模型，（e）溶液X-射线散射和用二十面体或球面谐波的数据分析，和（f）用质谱分析所得多肽的蛋白水解敏感性研究。 适用于时间分辨分析的系统包括CCMV和RYMV的可逆溶胀和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cryoEM（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。 我们已经选择了模型系统描述他们的实验可访问性，但预计他们的研究，在所描述的上下文中，将提供一个一般和基本的理解蛋白质多态性的原则和能力，专门改变这种转变。

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.