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Biophys. J. BioFAST: First Published April 1, 2005. doi:10.1529/biophysj.105.059170
© 2005 by the Biophysical Society.

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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Unraveling protein-protein interactions in living cells with fluorescence fluctuation brightness analysis

Yan Chen 1, Li-Na Wei 1 and Joachim D. Müller 1*

1 University of Minnesota

* To whom correspondence should be addressed. E-mail: mueller{at}physics.umn.edu.

Submitted on January 6, 2005
Revised on March 7, 2005
Accepted on 28 March 2005


  Abstract
Fluorescence correlation spectroscopy is a potentially powerful tool for measuring protein-protein interactions directly in single living cells. We previously reported on the detection of homodimer formation in cells using molecular brightness analysis. Here, we extend the technique to detect binding between different proteins. Proteins are labeled with the fluorescent markers YFP and CFP. We first determine the co-expression ratio of both proteins by measuring the intensity ratio with a dual-color setup. The effect of FRET on the intensity ratio is explicitly taken into account. The brightness of cells co expressing both proteins is measured in a single-color setup. Selecting the laser wavelength of the two-photon light source allows us to coexcite both proteins, or to selectively excite YFP labeled proteins. This approach enables us to distinguish between homodimer and heterodimer formation. We first present the theory and then demonstrate experimental feasibility using the ligand binding domains of retinoic acid receptor (RARLBD) and of retinoid X receptor (RXRLBD). Both proteins form heterodimers, and RXRLBD also forms homodimers in the presence of its agonist. We explore binding between these proteins in the presence and absence of RXR agonist. Our results demonstrate that brightness analysis offers a quantitative method for determining protein interactions in cells.

Key Words: FCS, fluorescence correlation spectroscopy, hetero-dimer, nuclear receptor, photon counting histogram, two-photon




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Copyright © 2005 by the Biophysical Society.