Measuring the Pore-Size and Dynamics of the MscL Mechanosensitive Channel using smFRET

Abstract

Mechanosensitive ion channels are membrane-bound proteins that let solutes (and water) flow in and out of the cell in response to membrane deformation. They are involved in sensory modalities, like touch, sound, fluid balance, and blood pressure. The mechanosensitive channel of large conductance (MscL) is to date the best studied mechanosensitive channel and serves as a model for channel mechanosensitivity. MscL has been crystallized in the closed form, but not in the open form. Some fundamental questions remain to date not fully answered including - what is the diameter of the open channel pore? Previously, several reports described open channel structure of MscL that was obtained by using either EPR or FRET spectroscopy on large ensembles of MscL channels. Although these studies provided good estimates of the open MscL pore, the size of the pore of a fully open channel still remains to be determined. Here we report a study describing both the open and closed states of MscL using single molecule fluorescence energy transfer (smFRET). We measured the distance changes of a number of residues from the three MscL alpha-helices TM1, TM2 and CP, i.e. I25, A27, and M42 on TM1; Y75 and I96 on TM2 and A110 and V120 on CP. Since we measured two or more sites on each alpha helix, rotational tilting angles, as well as the translational movements of the helices could be obtained. From these measurements, a structure of the protein in the open state was determined, using the crystal structure of the closed channel as a reference. From the obtained open structure we could determine the pore size of the fully open channel. To understand the channel mechanosensitivity, we investigated the dynamics of the conformational change of the channel.