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Biochem J

Title:		Protein interaction quantified in vivo by spectrally resolved fluorescence resonance energy transfer
Author(s):		Raicu V; Jansma DB; Miller RJ; Friesen JD;
Address:		"Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada. vraicu@uwm.edu"
Journal Title:		Biochem J
Year:		2005
Volume:		385
Issue:		Pt 1
Page Number:		265 - 277
DOI:		10.1042/BJ20040226
ISSN/ISBN:		1470-8728 (Electronic) 0264-6021 (Print) 0264-6021 (Linking)
Abstract:		"We describe a fluorescence resonance energy transfer (FRET)-based method for finding in living cells the fraction of a protein population (alpha(T)) forming complexes, and the average number (n) of those protein molecules in each complex. The method relies both on sensitized acceptor emission and on donor de-quenching (by photobleaching of the acceptor molecules), coupled with full spectral analysis of the differential fluorescence signature, in order to quantify the donor/acceptor energy transfer. The approach and sensitivity limits are well suited for in vivo microscopic investigations. This is demonstrated using a scanning laser confocal microscope to study complex formation of the sterile 2 alpha-factor receptor protein (Ste2p), labelled with green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP respectively), in budding yeast Saccharomyces cerevisiae. A theoretical model is presented that relates the efficiency of energy transfer in protein populations (the apparent FRET efficiency, E(app)) to the energy transferred in a single donor/acceptor pair (E, the true FRET efficiency). We determined E by using a new method that relies on E(app) measurements for two donor/acceptor pairs, Ste2p-CFP/Ste2p-YFP and Ste2p-GFP/Ste2p-YFP. From E(app) and E we determined alpha(T) approximately 1 and n approximately 2 for Ste2 proteins. Since the Ste2p complexes are formed in the absence of the ligand in our experiments, we conclude that the alpha-factor pheromone is not necessary for dimerization"
Keywords:		Bacterial Proteins/*chemistry/genetics/*metabolism *Fluorescence Resonance Energy Transfer Green Fluorescent Proteins/*chemistry/genetics/*metabolism Luminescent Proteins/*chemistry/genetics/*metabolism Photobleaching Protein Binding Saccharomyces cerevis;
Notes:		"MedlineRaicu, Valerica Jansma, David B Miller, R J Dwayne Friesen, James D eng England 2004/09/09 Biochem J. 2005 Jan 1; 385(Pt 1):265-77. doi: 10.1042/BJ20040226"