FRET imaging of protein-protein interactions inside living cells

National Institute On Alcohol Abuse And Alcoholism

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Abstract

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) To develop a method for simultaneously monitoring two independent protein-protein interactions inside cells, and to use this approach to simultaneously monitor CaMKII activation and T-site ligand binding, as well as to generate and monitor binary biosensors. 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, Dr. Alexandra Olaya-Castro to study the theoretical basis for strong coupling in fluorescent proteins, and Dr. Prem Kumar to study photon entanglement 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.