Polarized Fluorescence Correlation Spectroscopy (pFCS): A Single-Molecule Method for Simultaneously Measuring Homo-FRET, Brightness, and the Diffusion of Protein Complexes in Living Cells

Abstract

FRET is a phenomenon where excited state energy of a fluorescent donor molecule is transferred, by a non-radiative dipole-dipole coupling mechanism, to a nearby acceptor. Because the efficiency of FRET provides information regarding the distance separating the donor and acceptor, FRET is used to study protein interactions in living cells. In conjunction with automated microscopy, FRET can also be used to screen for drugs that perturb specific protein-protein interactions. The utility of FRET, however, is limited by the possibility of both false positive (caused by over expression and molecular crowding), and by false negative signals (resulting from separation distances greater than 10 nm and from very low dipole orientation factors). To overcome these limitations we have developed Polarized Fluorescence Correlation Spectroscopy (pFCS), a single-molecule based method to characterize the interactions of proteins in complexes. Using pFCS, Homo-FRET (a 1-10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and the lateral diffusion coefficient (an attribute sensitive to viscosity, mass, and the shape of a protein complex) can be simultaneously measured. With these measurements, the interpretation of FRET can be rigorously constrained thus reducing the likelihood of both false negative and false positive interpretations. Standards consisting of tandem covalently linked concatemers of between 1 and 6 Venus molecules were used to validate pFCS in both solution and in cells. The utility of pFCS was demonstrated by measuring differences in Homo-FRET, subunit stoichiometry and correlation time between Venus-tagged CaM-kinase-II holoenzyme (α or β).