Determining the stoichiometry of protein heterocomplexes in living cells with fluorescence fluctuation spectroscopy

doi:10.1073/pnas.0606557104

Determining the stoichiometry of protein heterocomplexes in living cells with fluorescence fluctuation spectroscopy

Yan Chen and
Joachim D. Müller*

School of Physics and Astronomy, University of Minnesota, 116 Church Street Southeast, Minneapolis, MN 55455

Edited by Roger Y. Tsien, University of California at San Diego, La Jolla, CA, and approved December 20, 2006 (received for review July 31, 2006)

Abstract

The brightness of fluorescence fluctuations provides information about protein interactions in the intercellular environment under equilibrium conditions. Here we demonstrate that the stoichiometry of a protein complex containing two proteins labeled with CFP and YFP can be determined by brightness analysis. The brightness profile, which characterizes the brightness as a function of the labeled protein coexpression ratio, together with brightness titration experiments, provides sufficient information to quantify the composition of a protein complex under stoichiometric binding conditions. The selective and simultaneous excitation of proteins labeled with CFP and YFP by choosing different excitation wavelengths is used to identify the composition of the protein complex. Interactions between nuclear receptors and their coactivators play a crucial role in the regulation of gene expression. We choose the ligand-binding domain of retinoic X receptor and the nuclear receptor interacting domain of the steroid receptor coactivator-1 as a model for exploring the formation of a hetero-oligomer by brightness analysis directly in living cells. Our results show the formation of a heterotetramer with three nuclear receptors binding to the coactivator domain. Elimination of one of the nuclear receptor binding sites through a truncation mutant changed the interaction between both proteins significantly and led to a nuclear receptor-induced oligomerization of the truncated coactivator. Quantifying protein interactions in a cell is an important step in understanding cellular function on a molecular level. This study provides proof-of-principle experiments that illustrate the potential of brightness analysis as a powerful tool for quantifying protein interactions in living cells.

Footnotes

*To whom correspondence should be addressed. E-mail: mueller{at}physics.umn.edu
Author contributions: Y.C. and J.D.M. designed research; Y.C. performed research; Y.C. and J.D.M. analyzed data; and J.D.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0606557104/DC1.
Abbreviations:

FFS,

fluorescence fluctuation spectroscopy;

RXR,

retinoic X receptor;

LBD,

ligand-binding domain;

SRC1,

steroid receptor coactivator 1;

NR,

nuclear receptor;

NID,

NR interaction domain;

9cRA,

9-cis-retinoic acid.