FRET imaging of protein-protein interactions inside living cells

细胞生物光子学部分的主要目的是使用成像和光谱技术，如双光子显微镜，光谱成像，荧光寿命显微镜，荧光相关光谱（FCS）和荧光各向异性分析来研究蛋白质复合物如何调节活细胞中的突触功能。最近，我们集中精力利用福斯特共振能量转移（FRET）来监测蛋白质-蛋白质相互作用。这种方法具有很大的潜力，研究蛋白质的相互作用，因为它是敏感的变化，在1-10纳米尺度上分离两个荧光团的距离。FRET成像结合绿色荧光蛋白（GFP）的光谱变体的发展提供了机会，以遗传标记感兴趣的突触蛋白质和监测其相互作用与其他标记的蛋白质在真实的时间。 目前，我们在实验室有5个项目： 第一个项目的目标是调整FPFA（荧光偏振和波动分析），各向异性寿命衰减分析和FCS的混合，以：监测蛋白质复合物结构的动态变化，以确定蛋白质-蛋白质相互作用的特定位点，并自动化FPFA，以实现对构成相互作用组的数百种潜在相互作用蛋白质之间的蛋白质-蛋白质相互作用的大规模生物物理分析（细胞分子相互作用的完整网络）。 我们的第二个项目旨在与Anne Kenworthy和Eric Long博士合作应用FPFA。 第三个项目涉及开发基于光纤的光谱学，用于监测活小鼠中表达的荧光探针。一旦完善，这些基于纤维的系统将用于监测转基因小鼠执行行为任务时特定神经元子集中表达的荧光团的特定变化。我们在第三个项目中的生物学目标是了解基底神经节如何在分子水平上控制自愿行为。我们的技术目标是采用光纤和先进的光谱技术来动态监测生物传感器和/或功能大脑深处神经元中的蛋白质-蛋白质相互作用。 我们的第四个项目旨在了解最近发现的荧光蛋白组装中观察到的超快能量转移的机制。 我们的第五个项目，在与保罗空白和Wieb货车德梅尔博士合作，是发展的基本理论，允许精确估计生物成分之间的距离的基础上FRET测量，尽管荧光团方向的不确定性。

The principal aim of the Section on Cellular Biophotonics is to use imaging and spectroscopy techniques, such as two-photon microscopy, spectral imaging, fluorescence lifetime microscopy, fluorescence correlation spectroscopy (FCS), and fluorescence anisotropy analysis to study how protein complexes regulate synaptic function in living cells. Recently, we have concentrated our efforts on utilizing Forster Resonance Energy Transfer (FRET) to monitor protein-protein interactions. This method has great potential for studying protein interactions because it is sensitive to changes in the distance separating two fluorophores on the 1-10 nm scale. FRET imaging in conjunction with the development of spectral variants of Green Fluorescent Protein (GFP) provides the opportunity to genetically tag synaptic proteins of interest and monitor their interactions with other labeled proteins in real time. Currently we have 5 projects in the lab: The goal of the first project is to adapt FPFA (fluorescence polarization and fluctuation analysis), a mixture of anisotropy lifetime decay analysis and FCS, to: monitoring dynamic changes in protein complex structure, to identify specific sites of protein-protein interactions, and to automate FPFA to enable the wide-scale biophysical analysis of protein-protein interactions between hundreds of potentially interacting proteins that comprise the interactome (a cells complete network of molecular interactions). Our second project is aimed at applying FPFA in collaborations with Drs. Anne Kenworthy and Eric Long. The third project is involved in developing fiber-optics based spectroscopy for use in monitoring fluorescent probes expresses in living mice. Once perfected, these fiber-based systems will be used to monitor specific changes in fluorophores expressed in specific subsets of neurons of transgenic mice as they perform behavioral tasks. Our biological goal in this third project is to understand how the basal ganglia controls voluntary action at the molecular level. Our technological goal is to adapt fiber optics and advanced spectroscopic techniques to dynamically monitor bio-sensors and/or protein-protein interaction in neurons deep within the functioning brain. Our fourth project is aimed at understanding the mechanism of recently discovered ultra-fast energy transfer observed in assemblies of fluorescent proteins. Our fifth project, in collaboration with Drs. Paul Blank and Wieb van der Meer, is to develop the underlying theory to allow accurate estimates of distance between biological components based on FRET measurements despite uncertainties in fluorophore orientation.