Cells respond to mechanical stimuli with the aid of mechanosensitive (MS) channels, a class of gated ion channels present in the cell membrane. Mechanosensitive Channel of Large Conductance (MscL) is one of the most studied bacterial MS channels. Since MscL couples the membrane tension directly to large conformational changes, following these changes with spectroscopic techniques would provide valuable information on mechanosensation at the molecular level.
Previously, the gradual MscL opening could be achieved by changing the hydrophobicity of the pore lining at a critical residue (G22) in one to five subunits. Here, based on this principle, we activated MscL into different intermediate states by generating a negative charge on the G22C position of defined number of subunits using a light-switch. Illumination with UV light photolyses the cysteine-attached label and leaves a carboxylic acid attached to the channel. We monitored the resulting structural changes by EPR. We generated the heteropentamers of MscL with a defined number of the light switch and EPR spin label per pentamer by (i) labeling different MscL populations with the light switch and EPR spin label, individually; (ii) obtaining the monomers of labeled homopentameric MscLs; (iii) mixing light-switch-labeled MscL and spin-labeled MscL monomers in different ratios, and (iv) re-associating MscLs back to pentameric channels. While the light switch provided a high spatiotemporal resolution, EPR provided the required sensitivity to detect subtle changes in the microenvironment of different positions on the protein.