Determination of supramolecular structure and spatial distribution of protein complexes in living cells

Abstract

Resonant energy transfer from an optically excited donor molecule to a non-excited acceptor molecule residing nearby is widely used to detect molecular interactions in living cells. To date, resonant energy transfer has been used to obtain stoichiometric information, such as the number of proteins forming a complex, for a handful of proteins, but only after performing sequential scans of the emission wavelengths, excitation wavelengths, or sometimes both. During this lengthy process of measurement, the molecular makeup of a cellular region may change, limiting the applicability of resonant energy transfer to the determination of cellular averages. Here, we demonstrate a method for the determination of protein complex size, configuration, and spatial distribution in single living cells. It relies on a spectrally resolved two-photon microscope, a simple but competent theory, and a judicious selection of fluorescent tags. This approach eventually may lead to tracking the dynamics of individual molecular complexes inside living cells. The combination of spectrally resolved two-photon microscopy, fluorescent tags and appropriate theory makes it possible to determine the complex size, configuration and spatial distribution of proteins in single living cells. The findings made could lead to ways of tracking the cellular dynamics of individual molecular complexes.