Association-Dissociation Dynamics of Transmembrane Helices as Detected by Single-Molecule Fluorescence Microscopy

Abstract

Conformational fluctuations of helical membrane proteins among active-inactive conformations are crucial for their functions. Not only the amino acid sequence of the protein but also the composition of surrounding lipids should significantly affect the fluctuation, although the fundamental principles are mostly unknown. Experimental systems using model transmembrane helices and lipid bilayers have been proposed to be useful for direct measurements of elementary processes that determine the folding and conformational change of membrane proteins, such as insertion of the helix into lipid bilayers and helix-helix interaction in the bilayers. Here we show that real-time detection of self-association and dissociation of the transmembrane helix (AALALAA)3 is possible by single molecule FRET detection using the Cy3B- and Cy5- labeled helices incorporated into large unilamellar vesicles (LUVs, diameter ∼100 nm) composed of POPC. The LUVs were fixed on a glass surface via biotin-avidin interaction and observed under a total internal reflection fluorescence microscope. After simultaneous observation of Cy3B and Cy5 fluorescence with 10-20-ms time resolution, LUVs that had incorporated only one Cy3B- and one Cy5- labeled helices were selected based on the number of photobleaching steps, and single-molecular FRET from Cy3B to Cy5 was analyzed. FRET time course revealed that the helices fluctuate between monomer and dimer with a time scale of subseconds in the presence of cholesterol in the LUVs. The above experimental system will be useful for measurement of kinetics of helix-helix interactions in membranes with various lipid compositions.