Single Molecule FRET Reveals Pore Size and Opening Mechanism of MscL

The mechanosensitive channel of large conductance, which serves as a model system for mechanosensitive channels, has previously been crystallized in the closed form, but not in the open form. Ensemble measurements and electrophysiological sieving experiments show that the open-diameter of the channel pore is >25{\AA}, but the exact size and whether the conformational change follows a helix-tilt or barrel-stave model are unclear. Here we report measurements of the distance changes on liposome-reconstituted MscL transmembrane {\alpha}-helices, using a "virtual sorting" single-molecule fluorescence energy transfer. We observed directly that the channel opens via the helix-tilt model and the open pore reaches 2.8 nm in diameter. In addition, based on the measurements, we developed a molecular dynamics model of the channel structure in the open state which confirms our direct observations.