PROTEIN POLYMORPHISM DYNAMICS IN ICOSAHEDRAL VIRUSES

我们建议继续我们对病毒颗粒中蛋白质多态的研究，并将其扩展到适用于时间分辨方法研究的第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，并确定使它们适合于多个四元结构构象的分子开关和化学表面特征。在每一种情况下，我们都已经或正在通过X射线结晶学确定至少一种形式的粒子的近原子分辨率结构。如果交替的多晶型由于中等非均相或不稳定而不能结晶，则从高分辨率结构导出的原子亚单位模型将被用来通过下面描述的方法来开发多晶型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，也可以作为重组组件(病毒样颗粒；VLP)在所使用的异源表达系统的细胞内组装。因此，我们从详细的建模和物理研究中得出的假设可以通过分子遗传改变和对随后的组装表型的分析直接进行验证。对苜蓿花叶病毒(AMV)、豌豆褪绿斑驳病毒(CCMV)、水稻黄斑驳病毒(RYMV)、鸡舍病毒(FHV)、绵羊夜蛾病毒(NomegaV)和LA病毒的病毒亚基静态多态性的研究将通过各种方法启动或扩展。这些方法包括(A)常规或超低分辨率X射线结晶学，(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，将高分辨率坐标的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派生模型进行改进，(E)溶液X射线散射和二十面体或球面谐和的数据分析，以及(F)利用质谱分析得到的多肽进行蛋白质降解敏感性研究。适合于时间分辨分析的体系包括CCMV和RYMV的可逆溶胀和NomegaV的大规模四元结构转变。这些将通过时间分辨的x射线散射(100毫秒制度)和低温电子显微镜(500毫秒制度)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，因为它们的实验可及性，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原理的一般和基本的理解，以及具体改变这种转变的能力。

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.