Fluorescence Fluctuation Spectroscopy of Ternary Protein Interactions in Living Cells

Abstract

Because many proteins contain multiple interaction sites, the formation of ternary or higher-order protein complexes are frequently encountered and play a critical role in many cellular processes. Fluorescence fluctuation spectroscopy (FFS) characterizes the brightness of fluorescently-labeled proteins, which provides a quantitative and noninvasive method to characterize protein interactions in living cells. Here we focus on extending two-photon brightness microscopy to the characterization of three interacting protein species, with each species carrying a differently colored tag. The fluorescence signal is split into three detection channels by optical filters. We discuss the choice of tags and filters that optimize the signal-to-noise ratio of resolving species, and the influence of spectral cross-talk on data analysis by time-integrated cumulants of the photon counts. While proof of principal experiments with dyes are useful, our main challenge remains the limited choice of fluorescence proteins that have simple photophysics and good color separation for cellular applications. We characterize different combinations of fluorescent proteins and evaluate their potential for three-color FFS studies of ternary protein interactions. This work has been supported by grants from the National Institutes of Health (GM64589) and the National Science Foundation (PHY 0346782).