Expression, purification, and reconstitution of small-conductance mechanosensitive channel into lipid bilayer: ready...

Solid-state NMR (ssNMR) provides unique advantages for resolving the structure and interactions of membrane proteins within native-mimicking lipid membranes. However, the challenge is preparing milligrams of stable, homogeneous proteoliposomes samples to achieve high-resolution data. Mechanosensitive channels of small conductance (MscS) are important members of the bacterial mechanosensitive channel family, functioning as emergency release valves to protect cells from osmotic lysis. In this study, AlphaFold was employed to predict the structures of MscS proteins from four bacterial species, shedding light on further experimental work. To obtain homogeneous MscS protein in high yield, several key parameters were systematically optimized, including the expression host strain, induction temperature, and optical density at induction. A "dual-media" approach was also applied. Pa-MscS demonstrated stable high-level expression, yielding about 30 mg/L using "dual-media" method. Gelfiltration chromatography and chemical crosslinking confirmed that Pa-MscS assembles homogeneously into a homoheptamer. Pa-MscS was successfully reconstituted into proteoliposomes, by optimizing the lipid composition of the liposomes, detergent removal conditions, and the phospholipid-to-protein ratio. Finally, 2D ¹⁵N-¹³Cα (NCA) spectroscopy of the Pa-MscS proteoliposomes revealed well-resolved spectra by ssNMR, enabling high-resolution structural studies. This integrative approach provides a robust framework for obtaining high-yield and homogeneous MscS sample for ssNMR-based functional and mechanistic studies, laying the groundwork for future investigations into MscS and therapeutic development.