Second-Generation SuptoxR Strains for High-Level Recombinant Membrane Protein Production.

is one of the most widely utilized hosts for recombinant protein production, including that of membrane proteins (MPs). We have recently engineered a specialized strain for enhanced recombinant MP production, termed SuptoxR. By appropriately co-expressing the effector gene , SuptoxR can suppress the high toxicity, which is frequently observed during the MP-overexpression process, and, at the same time, enhance significantly the cellular accumulation of membrane-incorporated and properly folded recombinant MP.

Pocket delipidation induced by membrane tension or modification leads to a structurally analogous mechanosensitive channel state.

The mechanosensitive ion channel of large conductance MscL gates in response to membrane tension changes. Lipid removal from transmembrane pockets leads to a concerted structural and functional MscL response, but it remains unknown whether there is a correlation between the tension-mediated state and the state derived by pocket delipidation in the absence of tension. Here, we combined pulsed electron paramagnetic resonance spectroscopy and hydrogen-deuterium exchange mass spectrometry, coupled with molecular dynamics simulations under membrane tension, to investigate the structural changes associated with the distinctively derived states.

In-Lipid Structure of Pressure-Sensitive Domains Hints Mechanosensitive Channel Functional Diversity.

The mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis has been used as a structural model for rationalizing functional observations in multiple MscL orthologs. Although these orthologs adopt similar structural architectures, they reportedly present significant functional differences. Subtle structural discrepancies on mechanosensitive channel nanopockets are known to affect mechanical gating and may be linked to large variability in tension sensitivity among these membrane channels.

Allosteric activation of an ion channel triggered by modification of mechanosensitive nano-pockets.

Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation. However, the effect of hindering lipid-chain penetration into nano-pockets on channel structure has not been demonstrated. Here we identify nano-pockets on the large conductance mechanosensitive channel MscL, the high-pressure threshold channel.

Optimization of Recombinant Membrane Protein Production in the Engineered Strains SuptoxD and SuptoxR.

Membrane proteins (MPs) execute a wide variety of critical biological functions in all living organisms and constitute approximately half of current targets for drug discovery. As in the case of soluble proteins, the bacterium has served as a very popular overexpression host for biochemical/structural studies of membrane proteins as well. Bacterial recombinant membrane protein production, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low levels of final biomass and minute volumetric yields.