Single Molecule FRET Analysis on Realtime SNARE Assembly

Abstract

Although it is well known that SNARE proteins drive membrane fusion by forming ternary SNARE complexes, the question that how more than one SNAREs cooperate with each other in time domain remains largely unanswered. To address this question directly, we modified single-molecule FRET technique to track complex formation of several SNAERE proteins simultaneously. This technique enables one to track many proteins in real-time thereby revealing novel property of protein-protein interaction, which is has not been accessible in single-molecule nor bulk FRET experiments. Furthermore, we used 3-color FRET technique by employing DNA based probe for vesicle content mixing to observe SNARE complex assembly and concomitant vesicle fusion simultaneously. This gives direct connection between number of SNARE complex and pore opening of vesicle.