Observing Nuclear Receptor / Coactivator Interactions in Live Cells by Hetero-Species Partition Analysis

Abstract

Measuring the binding curve and stoichiometry of protein complexes in living cells is a prerequisite for quantitative modeling of cellular processes. Dual-color fluorescence fluctuation spectroscopy provides a general framework for detecting protein interactions. However, quantitative characterization of protein hetero-interactions remains a difficult task. To address this challenge we introduce hetero-species partition (HSP) analysis for measuring protein hetero-interactions of the type D + nA -> DAn. HSP directly identifies the hetero-interacting species from the sample mixture and determines the binding curve and stoichiometry in the cellular environment. The method is applied to measure the ligand-dependent binding curve of the nuclear receptor retinoic X receptor to the coactivator transcription intermediate factor 2. The binding stoichiometry of this protein system has not been directly measured yet. A previous study using protein fragments observed a higher binding stoichiometry than biologically expected. We address this difference in stoichiometry by measuring the binding curves of the full-length proteins in living cells. This study provides proof-of-principle experiments that illustrate the potential of HSP as a general and robust analysis tool for the quantitative characterization of protein hetero-interactions in living cells.