FRET imaging of protein-protein interactions inside living cells

细胞生物光子学部分的目标是发展新形式的显微镜和光度学来研究生理条件下蛋白质与蛋白质的相互作用。我将在这一节概述四个主要项目的现状。 渣打银行有四个具体目标： 1)自动化荧光偏振和波动分析(FPFA)显微技术，并将该技术应用于CaMKII激活和T位点配体相互作用等生物学问题。 2)发展一种同时监测细胞内两个独立的蛋白质-蛋白质相互作用的方法，并利用该方法产生二元生物传感器。 3)研究荧光蛋白之间的超快能量转移机制，了解荧光蛋白之间相干激子相互作用的基础。 4)发展一种利用脑深部纤维光度法监测蛋白质相互作用的方法。 具体目标1在阮博士的指导下，我们设计并制造了一台自动化显微镜，能够在一夜之间从玻璃底96孔板中的96个样品中收集FPFA数据。该仪器对所有的样品井都有很好的重复性，实验方差很小。我们正在与范德比尔特大学的安妮·肯沃斯博士实验室合作，展示这种自动化仪器的实用性。 具体目标2该项目主要在阮氏博士的指导下进行，并得到普赫勒大学的协力支持。Tuan已经证明了同源FRET和异源FRET可以同时被测量，并且这些测量可以跟随同源FRET和异源FRET对的接近程度的独立变化。Tuan通过使用Hetero-FRET同时监测CaM-Kinase-II T位点配体的结合，同时使用Homo-FRET测量配体触发的激酶全酶结构的构象变化，证明了这种方法的实用性。Puhl博士目前正在开发同时监测低亲和力和高亲和力的游离钙浓度的二元生物传感器，以进一步展示这种方法的实用性。 具体目标3 Kim博士一直在使用时间分辨各向异性、FCS和反聚束来开发新的分析和仪器来研究荧光蛋白质之间的超快(快于140 ps)能量转移。我们正在与加州大学圣克鲁斯分校的陈博士和克里格博士合作，使用CD光谱来检测成对荧光蛋白之间的相干相互作用。我们的结果表明，相干的能量转移机制是导致这种意外的高速能量转移的原因。我们推测，这种机制在发展量子计算机方面可能会有用处。 具体目标4与Lovinger博士的实验室(LIN)合作，Nguyen博士和Kim博士正在开发基于光纤的仪器，以监测基于FRET的生物传感器在活体小鼠中cAMP和A-激酶活性的基础上监测荧光寿命的变化。

The objective of the Section on Cellular Biophotonics is to develop new forms of microscopy and photometry to study protein-protein interactions under physiological conditions. I will outline the current status of the four major projects in the section. SCB has four Specific Aims: 1) To automate fluorescent polarization and fluctuation analysis (FPFA) microscopy, and to apply this technology to biological problems such as CaMKII activation and T-site ligand interactions. 2) To develop a method for simultaneously monitoring two independent protein-protein interactions inside cells, and to use this approach to generate binary biosensors. 3) To investigate the mechanism of ultrafast energy transfer between fluorescent proteins and understanding the basis for coherent excitonic interactions between fluorescent proteins. 4) To develop a methodology for monitoring protein-protein interactions using deep-brain fiber photometry. Specific Aim 1 Under the direction of Dr. Nguyen, we have designed and built an automated microscope that is capable or collecting FPFA data from 96 samples in a glass-bottom 96-well plate overnight. This instrument has excellent repeatability across all sample wells, and the experimental variance is very low. We are collaborating with Dr. Anne Kenworthys laboratory at Vanderbilt University to demonstrate the utility of this automated instrumentation. Specific Aim 2 This project is primarily under the direction of Dr. Nguyen, with collaborative support of the Puhl. Tuan has demonstrated that homo-FRET and hetero-FRET can be measured simultaneously and that these measurements can follow independent changes in the proximity of homo-FRET and hetero-FRET pairs. Tuan has demonstrated the utility of this approach by simultaneously monitoring the binding of a CaM-Kinase-II T-site ligand using hetero-FRET while simultaneously measuring a conformational change in the kinase holoenzyme structure triggered by the ligand using homo-FRET. Dr. Puhl is currently developing binary biosensors that simultaneously monitor free calcium concentration with both low and high affinity to further demonstrate the utility of this approach. Specific Aim 3 Dr. Kim has been using time-resolved anisotropy, FCS, and antibunching to develop new analysis and instrumentation to investigate ultra-fast (faster than 140 ps) energy transfer between fluorescent proteins. We are collaborating with Drs. Chen and Kliger at UC Santa Cruz to use CD spectroscopy to detect coherent interactions between paired fluorescent proteins. Our results indicate that a coherent energy transfer mechanism is responsible for this unexpected high-speed energy transfer. We speculate that such a mechanism might have utility in developing quantum computers. Specific Aim 4 In collaboration with Dr. Lovinger's laboratory (LIN), Drs. Nguyen and Kim are developing fiber optic based instrumentation to monitor FRET-based biosensors for cAMP and A-kinase activity in living mice based on monitoring changes in fluorescence lifetime.