Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

Significance Understanding the working mechanism of membrane proteins is difficult even when crystal structures are available. One promising approach is ion mobility–mass spectrometry (IM-MS) that detects not only the mass-to-charge ratio but also the area of proteins by measuring the rotationally averaged collision cross-sections (CCS) in the gas phase. We identified detergents that allow the release of membrane proteins at low levels of collisional activation for native MS, thus avoiding denaturing effects. We studied the gating mechanism of an ion channel, which occurs through large conformational changes. Ability to detect several coexisting states during gating with a change as small as 3% will open new avenues for studying dynamic structures of membrane proteins. Mechanosensitive ion channels are sensors probing membrane tension in all species; despite their importance and vital role in many cell functions, their gating mechanism remains to be elucidated. Here, we determined the conditions for releasing intact mechanosensitive channel of large conductance (MscL) proteins from their detergents in the gas phase using native ion mobility–mass spectrometry (IM-MS). By using IM-MS, we could detect the native mass of MscL from Escherichia coli, determine various global structural changes during its gating by measuring the rotationally averaged collision cross-sections, and show that it can function in the absence of a lipid bilayer. We could detect global conformational changes during MscL gating as small as 3%. Our findings will allow studying native structure of many other membrane proteins.

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