PROTEIN POLYMORPHISM DYNAMICS IN ICOSAHEDRAL VIRUSES

我们建议继续研究病毒颗粒中的蛋白质多态性，并将其扩展到可通过时间分辨方法进行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，使它们适合多个四级结构构象。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。如果交替多晶不能结晶，因为它们是中度异质或不稳定的，那么从高分辨率结构衍生的原子亚基模型将用于开发多晶的伪原子分辨率结构，方法如下所述。我们正在研究的所有系统都可以在体外组装或作为重组组装（病毒样颗粒；VLPs）在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随后的组装表型分析直接验证。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）、芥蓝病毒（NomegaV）和LA病毒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为低分辨率实验密度函数，(d)通过分子力学和能量最小化来改进导出模型，(e)溶液x射线散射和使用二十面体或球面谐波进行数据分析。(f)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体系包括CCMV和RYMV的可逆膨胀和NomegaV的大规模第四纪结构转变。这些将通过时间分辨溶液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。我们选择了模型系统，因为它们的实验可及性，但预计他们的研究，在描述的背景下，将提供对蛋白质多态性原理的一般和基本的理解，以及特异性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.