FRET imaging of protein-protein interactions inside living cells

活细胞内蛋白质-蛋白质相互作用的 FRET 成像

• 批准号: 9551240
• 负责人: Steven S Vogel
• 金额: $ 132.9万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9551240
• 关键词: Advisory Committees; Affinity; Anisotropy; Binding; Biological; Biophotonics; Biosensor; Brain; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcium; Cell physiology; Cells; Collaborations; Cyclic AMP; Data Analyses; Energy Transfer; Fiber; Fiber Optics; Fluorescence; Fluorescence Resonance Energy Transfer; Glass; Holoenzymes; Homo; Human; Image; Individual; Institutes; Laboratories; Ligands; Logistics; Manuscripts; Measurement; Measures; Mediating; Methodology; Methods; Microscope; Microscopy; Mission; Molecular; Molecular Conformation; Monitor; Mus; Nanotechnology; National Institute on Alcohol Abuse and Alcoholism; Phosphotransferases; Photometry; Physiologic pulse; Physiological; Physiology; Presynaptic Terminals; Production; Proteins; Research; Sampling; Site; Spectrum Analysis; Speed; Structure; Synapses; Techniques; Technology; Translations; United States National Institutes of Health; Universities; base; calmodulin-dependent protein kinase II; design; imaging modality; imaging probe; instrument; instrumentation; interest; live cell microscopy; neuronal cell body; programs; protein complex; protein protein interaction; quantum computer; synaptic function; time use; tool development

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. 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. 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. A manuscript describing this instrumentation is being prepared. 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, antibunching, and paired-pulse correlation to develop new analysis and instrumentation to investigate ultra-fast (less than a few picoseconds) energy transfer between fluorescent proteins. His 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. Lovingers 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.

细胞生物光谱学部分的目的是开发新形式的显微镜和光度法，以研究生理条件下的蛋白质 - 蛋白质相互作用。我将概述本节中四个主要项目的当前状态。 SCB具有四个具体目标： 1）自动化荧光极化和波动分析（FPFA）显微镜，并将该技术应用于生物学问题。 2）开发一种同时监测细胞内部两种独立蛋白质蛋白质相互作用的方法，并使用这种方法来产生二元生物传感器。 3）研究荧光蛋白之间超快能量转移的机制。 4）开发一种使用深脑纤维光度法监测蛋白质蛋白相互作用的方法。 在Nguyen博士的指导下，我们设计并构建了一个自动显微镜，该显微镜能够或从96个玻璃底96孔板中的96个样品中收集FPFA数据过夜。该仪器在所有样品井中具有出色的可重复性，实验方差非常低。正在准备描述这种仪器的手稿。我们正在与范德比尔特大学的Anne Kenworthys实验室合作，以展示这种自动化仪器的实用性。 具体目的2该项目主要是在Nguyen博士的指导下进行的，并在PUHL的协作支持下。 TUAN证明可以同时测量Homo-Fret和Hetero-Fret，并且这些测量值可以遵循Homo-Fret和Hetero-Fret Pairs的接近性的独立变化。 Tuan通过同时使用异孔监测Cam-激酶-II T位配体的结合，同时测量了使用HOMO-FRET的配体触发的激酶Holoenzyme结构的构象变化，从而证明了这种方法的实用性。 PUHL博士目前正在开发二元生物传感器，这些生物传感器同时监测具有低亲和力的钙浓度，以进一步证明这种方法的实用性。 特定的目标3 Kim博士一直使用时间分辨的各向异性，FCS，抗启动和配对脉冲相关性来开发新的分析和仪器，以研究荧光蛋白之间的超快速（小于几次皮秒）能量转移。他的结果表明，连贯的能量传递机制是导致这种意外的高速能量转移的原因。我们推测这种机制可能在开发量子计算机方面具有实用性。 特定的目标4与Lovingers Laboratory博士（Lin）合作，Drs。 Nguyen和Kim正在开发基于光纤的仪器，以根据监测荧光寿命的监测变化，监测活小鼠中的基于FRET的生物传感器和A-激酶活性。