SOLUTION SAXS STUDIES OF CONFORMATIONAL CHANGES IN MEMBRANE FUSION AAA+ ATPASES

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。NSF和VCP是两种六聚三磷酸腺苷酶，在细胞膜内运输中起重要作用。脂质双分子层的融合是由存在于对立膜上的蛋白质介导的，这些蛋白质被称为SNAREs，它们结合在一起形成高度稳定的复合物。NSF利用ATP水解的能量来分解SNARE复合物，这样这些成分就可以被回收用于后续的融合。我们拟在NSF和VCP上进行溶液SAXS，研究ATP结合、水解和释放过程中的构象变化。其目的是：1)利用单个结构域的x射线结构生成NSF的atp结合、adp结合和无核苷酸状态的低分辨率模型；2)测量散射随时间的变化以获得动力学信息。通过将SAXS数据与我们实验室正在进行的其他生物物理和晶体学研究相结合，我们希望了解核苷酸水解和交换如何与NSF的结构变化和SNARE复合物的拆卸相结合。

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. NSF and VCP are two hexameric ATPases that play an essential role in intracellular membrane trafficking. Fusion of lipid bilayers is mediated by proteins present on opposing membranes, known as SNAREs, that associate to form a highly stable complex. NSF uses the energy of ATP hydrolysis to disassemble SNARE complexes so that the components can be recycled for subsequent rounds of fusion. We propose to carry out solution SAXS on NSF and VCP to study conformational changes upon ATP binding, hydrolysis and release. The aims are to 1) generate low resolution models of NSF in its ATP-bound, ADP-bound, and nucleotide-free states using the x-ray structures of the individual domains, and 2) measure changes in scattering as a function of time to obtain kinetic information. By combining the information derived from SAXS data with other biophysical and crystallographic studies underway in our laboratory, we hope to gain an understanding as to how nucleotide hydrolysis and exchange is coupled to the structural changes in NSF and disassembly of SNARE complexes.