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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。利用高静水压力和双光子荧光显微镜研究病毒组装。最近的研究发现，利用压力来评估多聚体蛋白、蛋白-核酸复合物和几种病毒的组装途径的中间状态，以了解成功组装所需的可塑性是如何被编码到衣壳蛋白亚基的折叠构象中。表征这些状态的兴趣在于它们在多大程度上与折叠和组装过程中存在的真正中间体相关。压力效应可以通过使用荧光光谱等光学方法来监测。利用压力促进病毒衣壳解离，我们已经检测到核糖核蛋白中间体的存在，其中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。由于压力可以用作“热力学镊子”，因此它与FCS技术的结合将成为监测病毒衣壳组装和拆卸以及评估这些过程中可能涉及的中间体信息的有力工具。

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.