Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel

By Yong Wang, Yanxin Liu, Hannah A DeBerg, Takeshi Nomura, Melinda Tonks Hoffman, Paul R Rohde, Klaus Schulten, Boris Martinac, and Paul R Selvin.

Published in eLife, 2014 Feb 18;3:e01834.
PMID: 24550255. PMCID: PMC3925968. Link to publication page.

Core Facility: Computational Modeling

Figure 1. A) Cartoon representation of the structure of MscL in the closed conformation in the (A) top view and (B) side view (PDB ID: 2OAR [Chang et al., 1998; Steinbacher et al., 2007]), and scheme of single molecule FRET setup.

MscL is a homo-pentamer consisting of five identical subunits. Each subunit consists of one cytoplasmic α-helix (CP) and two transmembrane α-helices (TM1 and TM2), which extend through the cell membrane and are joined by a periplasmic loop (Chang et al., 1998). (C) Residues measured using smFRET. Three residues on each of the transmembrane helices (M42C, A27C and I25C on TM1; Y75C, Q80C and V82C on TM2) were chosen. Note that no residues on the CP were chosen because the complete deletion of the CP does not change the gating parameters substantially (Anishkin et al., 2003). (D) Labeled MscL proteins were reconstituted into liposomes, which were then immobilized on a coverslip and used for smFRET experiments. (E) The addition of LPC traps the protein in the open conformation (Perozo et al., 2002b).


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 Å, 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 α-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.


Figure 2. The opening models for MscL.

Figure 4. Movement of residues.

Figure 7. Model of the MscL structure in the open conformation.