FRET imaging of protein-protein interactions inside living cells

细胞生物光子学部分的目标是开发新的显微镜和光度法来研究生理条件下蛋白质-蛋白质相互作用。我将在这一节中概述四大项目的现状。

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.