Fluorescence resonance energy transfer: a test-bench for fundamental laws of nature in vivo

Valerică Raicu; David B. Jansma; Kristin Michalski

doi:10.1117/12.659552

21 February 2006 Fluorescence resonance energy transfer: a test-bench for fundamental laws of nature in vivo

Valerică Raicu, David B. Jansma, Kristin Michalski

Proceedings Volume 6088, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV; 60880A (2006) https://doi.org/10.1117/12.659552
Event: SPIE BiOS, 2006, San Jose, California, United States

Abstract

Fluorescence Resonance Energy Transfer (FRET) - a process of nonradiative energy transfer from an optically excited molecule (donor, D) to an unexcited nearby molecule (acceptor, A) - is a powerful tool in studies of protein-protein interactions in living cells. FRET can be quantified, e.g., by measuring an increase in the donor fluorescence after inactivating the acceptor through photobleaching. In spite of its sheer simplicity, this method also introduces donor bleaching, which often complicates the interpretation of data. Correction methods are complicated by the fact that D photobleaching depends on whether a D molecule is free or coupled to an A molecule. In this communication we show that, instead of being a nuisance, donor bleaching actually can be harnessed to provide invaluable information about a population of interacting proteins in vivo. We present data on proteins tagged with fluorescent molecules, which indicate that donor and acceptor bleaching kinetics reveal quantitative information on the stoichiometry of proteinprotein interaction. Under appropriately chosen conditions, we are able to model the bleaching kinetics of D and A (both interacting and noninteracting) and determine the stoichiometry of the protein interaction. This provides a method for imaging protein complexes in living cells, which opens a way for testing the law of mass action in vivo.

Citation Download Citation

Valerică Raicu, David B. Jansma, and Kristin Michalski "Fluorescence resonance energy transfer: a test-bench for fundamental laws of nature in vivo", Proc. SPIE 6088, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV, 60880A (21 February 2006); https://doi.org/10.1117/12.659552

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KEYWORDS

Fluorescence resonance energy transfer

Molecules

Luminescence

Proteins

Green fluorescent protein

In vivo imaging

Energy efficiency

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