FRET imaging of protein-protein interactions inside living cells

细胞生物光子学部分的主要目的是利用成像和光谱学技术，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体如何调节活细胞的突触功能。最近，我们集中精力利用Forster共振能量转移(FRET)来监测蛋白质之间的相互作用。这种方法在研究蛋白质相互作用方面具有很大的潜力，因为它对1-10 nm尺度上两个荧光团之间的距离变化很敏感。FRET成像结合绿色荧光蛋白(GFP)光谱变体的开发，为从基因上标记感兴趣的突触蛋白并实时监测它们与其他标记蛋白的相互作用提供了机会。 目前我们在实验室中有5个项目： 第一个项目的目标是使荧光偏振和波动分析(FPFA)，即各向异性寿命衰减分析和FCS的混合体，用于：监测蛋白质复杂结构的动态变化，确定蛋白质-蛋白质相互作用的特定位置，并使荧光偏振和波动分析自动化，从而能够对组成相互作用组(一个完整的分子相互作用网络)的数百个潜在相互作用蛋白质之间的蛋白质-蛋白质相互作用进行广泛的生物物理分析。 我们的第二个项目是在与安妮·肯沃西博士和埃里克·朗博士的合作中应用FPFA。 第三个项目是开发基于光纤的光谱学，用于监测荧光探针在活体小鼠中的表达。一旦完善，这些基于纤维的系统将被用来监测转基因小鼠在执行行为任务时特定神经元亚群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在荧光蛋白质组装中观察到的超快能量转移的机制。 我们与Paul Blank博士和Wieb van der Meer博士合作的第五个项目是开发基本理论，以便在荧光团定位不确定的情况下，基于FRET测量准确估计生物成分之间的距离。

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.