USING 2-PHOTON FCS TO STUDY PRESSURE DISSOCIATION OF LARGE MULTIMERIC COMPLEXES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。用高静水压和双光子荧光显微镜研究病毒组装。最近在利用压力来评估多聚体蛋白质、蛋白质-核酸复合体和几种病毒的组装途径的中间状态方面出现了新的发现，试图了解成功组装所需的可塑性是如何编码成衣壳蛋白亚基的折叠构象的。人们对表征这些状态的兴趣在于它们与折叠和组装过程中存在的真正中间体的关联程度。压力效应可以通过荧光光谱等光学方法进行监测。利用压力促进病毒衣壳的解离，我们检测到核糖核蛋白中间体的存在，在那里外壳蛋白部分展开，但与RNA结合。这些中间体是抗病毒化合物的潜在靶点。由于压力可以被用作“热力学镊子”，它与FCS技术的结合将是监测病毒衣壳组装和拆解以及评估这些过程中可能涉及的中间体的信息的有力工具。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Study of the virus assembly using high hydrostatic pressure and 2 photon fluorescence microscopy. Recent findings have emerged on the use of pressure to assess intermediate states of multimeric proteins, protein-nucleic acid complexes and in the assembly pathways of several viruses, in an effort to understand how the plasticity required for successful assembly is coded into the folded conformation of a capsid protein subunit. Interest in characterizing these states lies in the extent to which they can be related to genuine intermediates present in folding and assembly processes. Pressure effects can be monitored by using optical methods such as fluorescence spectroscopy. Using pressure to promote viral capsids dissociation, we have detected the presence of ribonucleoprotein intermediates, where the coat protein is partially unfolded but bound to RNA. These intermediates are potential targets for antiviral compounds. Since pressure can be used as a "thermodynamic tweezer", its association with the FCS techniques will be a powerful tool to monitor the assembly and disassembly of virus capsids and to assess information on the possible intermediates involved in these processes.