Unraveling Ternary Protein Interactions in the Living Cell with Tricolor Fluorescence Microscopy

Abstract

Characterization of protein-protein interactions inside the living cell by fluorescence microscopy has been in almost every case limited to the study of pairwise interactions. Since most protein complexes contain more than two protein species, more powerful characterization methods than currently available are needed. We recently demonstrated the characterization of ternary protein systems in live cells using tricolor fluorescence fluctuation spectroscopy (FFS) combined with heterospecies partition analysis. The original description of tricolor FFS was based on a graphical interpretation of heterospecies brightness graphs. Here we advance the method by combining the original qualitative interpretation of tricolor FFS data with quantitative modeling to identify the binding affinity of ternary protein systems. We apply the method to the ternary protein system consisting of coactivator transcription intermediate factror2 and two nuclear receptors (retinoic acid receptor and retinoid X receptor) both in the absence and presence of the agonist. The results of the tricolor analysis were used to develop a potential interaction model for this protein system, and the influence of the agonist on the interactions will be discussed. This work has been supported by a grant from the National Institutes of Health (R01 GM64589).