FRET Spectrometry: A New Tool for the Determination of Protein Quaternary Structure in Living Cells

Förster resonance energy transfer (FRET) is an exquisitely sensitive method for detection of molecular interactions and conformational changes in living cells. The recent advent of fluorescence imaging technology with single-molecule (or molecular-complex) sensitivity, together with refinements in t...

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Bibliographic Details
Published in:Biophysical journal 2013-11, Vol.105 (9), p.1937-1945
Main Authors: Raicu, Valerică, Singh, Deo R.
Format: Article
Language:English
Subjects:
Biophysical Review
Cell Survival
Cells
energy transfer
Fluorescence
Fluorescence Resonance Energy Transfer
image analysis
Mass spectrometry
Molecular structure
protein quaternary structure
Protein Structure, Quaternary
protein subunits
protein transport
Proteins
Proteins - chemistry
receptors
Saccharomyces cerevisiae - cytology
spectroscopy
stoichiometry
transporters
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Summary:Förster resonance energy transfer (FRET) is an exquisitely sensitive method for detection of molecular interactions and conformational changes in living cells. The recent advent of fluorescence imaging technology with single-molecule (or molecular-complex) sensitivity, together with refinements in the kinetic theory of FRET, provide the necessary tool kits for determining the stoichiometry and relative disposition of the protomers within protein complexes (i.e., quaternary structure) of membrane receptors and transporters in living cells. In contrast to standard average-based methods, this method relies on the analysis of distributions of apparent FRET efficiencies, Eapp, across the image pixels of individual cells expressing proteins of interest. The most probable quaternary structure of the complex is identified from the number of peaks in the Eapp distribution and their dependence on a single parameter, termed pairwise FRET efficiency. Such peaks collectively create a unique FRET spectrum corresponding to each oligomeric configuration of the protein. Therefore, FRET could quite literally become a spectrometric method—akin to that of mass spectrometry—for sorting protein complexes according to their size and shape.
ISSN:0006-3495
1542-0086
DOI:10.1016/j.bpj.2013.09.015