FRET and Excitonic imaging of protein-protein interactions inside living cells

生物光子学和量子生物学实验室的目标是开发新形式的显微镜和光度学，以研究生理条件下的蛋白质-蛋白质相互作用。在这里，我们概述了LBQB三个主要项目的现状。 LBQB有三个具体目标： 第一章 利用Binary-FRET，我们的方法用于同时监测细胞内两种独立的蛋白质-蛋白质相互作用。 我们正在使用这种方法来同时监测CaMKII激活和T-位点配体结合，研究CaMKII易位到树突棘，以及研究与谷氨酸转运相关的EAAT-2的构象变化。 （二） 研究荧光蛋白间超快能量转移的机制，了解荧光蛋白间相干激子相互作用的基础。 第三章 建立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该项目主要由Nguyen博士指导，并得到Puhl博士的合作支持。Nguyen博士已经证明，homo-FRET和hetero-FRET可以同时测量，并且这些测量可以跟随homo-FRET和hetero-FRET对接近度的独立变化。Nguyen博士通过使用hetero-FRET同时监测CaM-激酶-II T位点配体的结合，同时使用homo-FRET测量与配体结合相关的激酶全酶结构的构象变化，证明了这种方法的实用性。除了生成支持Nguyen博士项目的结构外，Puhl博士目前还在开发二元生物传感器，该传感器可以同时监测低亲和力和高亲和力的游离钙浓度，以进一步证明这种方法的实用性。 具体目标2 Taumoefolau女士在她的论文项目中，与Kim博士（萨里大学）和Blank博士（NICHD）合作，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，以研究荧光蛋白之间的超快（超过140 ps）能量转移。我们还与加州大学圣克鲁斯的Chen和Kliger博士合作，使用CD光谱检测配对荧光蛋白之间的相干相互作用，Elisabetta Collini博士使用2D超快光谱研究红色荧光蛋白动力学，并与Alexandra Olaya-Castro博士研究荧光蛋白强耦合的理论基础。我们的研究结果表明，一个相干的能量转移机制是负责这种意想不到的高速能量转移。我们推测，这种机制可能在开发量子计算机方面具有实用性。 具体目标3 Nguyen博士与Lovinger博士的实验室（LIN）和Kim博士的实验室（萨里大学）合作，正在开发基于光子效率的光纤仪器，以监测基于FRET的生物传感器在活体小鼠中的cAMP和A-激酶活性，其基础是监测荧光寿命的变化。

The objective of the Laboratory of Biophotonics and Quantum Biology is to develop new forms of microscopy and photometry to study protein-protein interactions under physiological conditions. Here we outline the current status of the three major projects in LBQB. LBQB has three Specific Aims: 1) Utilize Binary-FRET, our method for simultaneously monitoring two independent protein-protein interactions inside cells. We are using this approach to simultaneously monitor CaMKII activation and T-site ligand binding, to study CaMKII translocation into dendritic spines, as well as for studying conformational changes in EAAT-2 associated with glutamate transport. 2) To investigate the mechanism of ultrafast energy transfer between fluorescent proteins and understanding the basis for coherent excitonic interactions between fluorescent proteins. 3) To develop a methodology for monitoring protein-protein interactions using deep-brain fiber photometry. Specific Aim 1 This project is primarily under the direction of Dr. Nguyen, with collaborative support from Dr. Puhl. Dr. Nguyen 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. Dr. Nguyen 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 associated with ligand binding using homo-FRET. In addition to generating constructs to support Dr. Nguyen's project, Dr. Puhl is also 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 2 Ms. Taumoefolau, in persuit of her thesis project, and in collaboration with Dr. Kim (University of Surrey) and Dr. Blank (NICHD) 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 also collaborating with Drs. Chen and Kliger at UC Santa Cruz to use CD spectroscopy to detect coherent interactions between paired fluorescent proteins, with Dr. Elisabetta Collini to use 2D ultra-fast spectroscopy to study red fluorescent protein dynamics, and with Dr. Alexandra Olaya-Castro to study the theoretical basis for strong coupling in 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 3 In collaboration with Dr. Lovinger's laboratory (LIN) and Dr. Kim's Laboratory (university of Surrey) Drs. Nguyen is developing photon-efficient 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.