Structural And Functional Exploration On Bacterial Mechanosensitive Channel Of Large Conductance (MscL)

The prokaryotic mechanosensitive channel of large conductance(Msc L)is a pressure-relief valve protecting the cell from lysing during acute osmotic downshock.Although many studies have focused on the structures and functions of Msc L since the identification of the protein,the following issues remain unresolved.Firstly,the influence of membrane environments on the structure and the molecular mechanism of Msc L remains unknown.All structures of Msc L were determined by X-ray crystallography in detergent environments,which possibly perturb the native structure of Msc L in membrane environments.Based on the X-ray crystal structure of Msc L,the "hydrophobic gate" mechanism was proposed.The structures and molecular mechanism of Msc L analyzed in the detergent environments need to be verified in native-like lipid bilayer environments.Secondly,previous electrophysiological experiments showed that brillant green(BG)can cause changes in Msc L channel activity,but the molecular mechanism,specific binding sites,and transport pathway remain unclear.Finally,Msc L could feel the cell membrane tension,and mechanical force sensitivities of Msc L molecules are different in different species.However,the key factors affecting mechanical force sensitivity require further investigations.To address the above issues,this thesis focused on the structure,function,and molecular mechanism of Msc L in the lipid bilayer by taking advantage of Solid-state NMR(ssNMR)spectroscopy,combining with biochemical experiments and molecular dynamic(MD)simulations.We have obtained the following results:1)The sample preparation conditions of Msc L in proteoliposomes of Ma-Msc L(Msc L from Methanosarcina acetivorans)were screened.2)The molecular mechanism of hydrophobic gating of Msc L in bilayers of E.coli lipid extracts was revealed by using a series of multi-dimensional ssNMR experiments.The residues(I21-T30)that form the hydrophobic gate in the channel of Msc L were identified by determining the 3D structure of Msc L and monitoring water accessibilities of the pore-line residues.Among these residues,residue F23 was identified by functional experiments as a key residue of the hydrophobic gate in the channel of Msc L.3)The binding sites of BG to Ma-Msc L were predicted by MD simulations,which were further verified by ssNMR experiments.The transport pathway of BG through the channel of Msc L was suggested as follows: M42→D38→K34→T30→ I27→F23→I17/P18.4)Key factors affecting mechanical force sensitivity were characterized by functional assays and ssNMR experiments of Ma-Msc L.The results showed that the lipid components of the membranes have little effect on mechanical force sensitivity,while the N-helix,loop region of Msc L significantly affects mechanical force sensitivity.In conclusion,the structure,gating mechanism,and function of Ma-Msc L in E.coli membranes were studied by ssNMR and functional experiments.The amino acids composition of hydrophobic gating and the hydrophobic gating mechanism were revealed by the 3D structure determinations and water accessibility analysis.Furthermore,the binding sites and the pathway for BG transport through the channel of Msc L were identified by MD simulations and ssNMR experiments.Finally,the factors affecting the mechanosensitive of Ma-Msc L were analyzed,and the key factors causing the poor mechanical force sensitivity of Ma-Msc L were explained.