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发展新的显微镜和光度学形式来研究生理条件下蛋白质-蛋白质的相互作用。在这里，我们概述了LBQB的三个主要项目的现状。 LBQB有三个具体目标： 1)利用二进制FRET，我们的方法可以同时监测细胞内两个独立的蛋白质-蛋白质相互作用。我们正在使用这种方法来同时监测CaMKII的激活和T位点的配体结合，研究CaMKII到树突棘的移位，以及研究与谷氨酸运输相关的EAAT-2的构象变化。 2)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。 3)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该项目主要在阮氏博士的指导下进行，并得到普尔博士的协力支持。Nguyen博士已经证明，可以同时测量同种FRET和异种FRET，并且这些测量可以跟随同种FRET和异种FRET对的接近程度的独立变化。Nguyen博士通过使用异源FRET同时监测CaM-Kinase-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，展示了这种方法的实用性。除了构建支持Nguyen博士的项目的结构外，Puhl博士目前还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2 Taumoefau女士，为了完成她的论文项目，并与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，以研究荧光蛋白质之间的超快(快于140 ps)能量转移。我们还与加州大学圣克鲁斯分校的Chen博士和Kriger博士合作，使用CD光谱检测成对荧光蛋白质之间的相干相互作用；与Elisabetta Collini博士合作，使用2D超快光谱研究红色荧光蛋白质动力学；以及与Alexandra Olaya-Castro博士合作，研究荧光蛋白质中强耦合的理论基础。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3与洛文杰博士的实验室(LIN)和金博士的实验室(萨里大学)合作，阮博士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cAMP和A-激酶活性的基础上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.