2,2,2-Trifluoroethanol Changes the Transition Kinetics and Subunit Interactions in the Small Bacterial Mechanosensitive Channel MscS

Abstract

2,2,2-Trifluoroethanol (TFE), a low-dielectric solvent, has recently been used as a promising tool to probe the strength of intersubunit interactions in membrane proteins. An analysis of inner membrane proteins of Escherichia coli has identified several SDS-resistant protein complexes that separate into subunits upon exposure to TFE. One of these was the homo-heptameric stretch-activated mechanosensitive channel of small conductance (MscS), a ubiquitous component of the bacterial turgor-regulation system. Here we show that a substantial fraction of MscS retains its oligomeric state in cold lithium-dodecyl-sulfate gel electrophoresis. Exposure of MscS complexes to 10-15 vol % TFE in native membranes or nonionic detergent micelles before lithium-dodecyl-sulfate electrophoresis results in a complete dissociation into monomers, suggesting that at these concentrations TFE by itself disrupts or critically compromises intersubunit interactions. Patch-clamp analysis of giant E. coli spheroplasts expressing MscS shows that exposure to TFE in lower concentrations (0.5-5.0 vol %) causes leftward shifts of the dose-response curves when applied extracellularly, and rightward shifts when added from the cytoplasmic side. In the latter case, TFE increases the rate of tension-dependent inactivation and lengthens the process of recovery to the resting state. MscS responses to pressure ramps of different speeds indicate that in the presence of TFE most channels reside in the resting state and only at tensions near the activation threshold does TFE dramatically speed up inactivation. The effect of TFE is reversible as normal channel activity returns 15-30 min after a TFE washout. We interpret the observed midpoint shifts in terms of asymmetric partitioning of TFE into the membrane and distortion of the bilayer lateral pressure profile. We also relate the increased rate of inactivation and subunit separation with the capacity of TFE to perturb buried interhelical contacts in proteins and discuss these effects in the framework of the proposed gating mechanism of MscS.

Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS

“…Upon exposure to TFE or HFIP, this intermediate state may be lost or collapse and as a result an off-diagonal spot is obtained. This is in agreement with the observation that TFE can have a stabilizing effect on the structure of globular proteins and affects the folding of such proteins [8,11]. In this scenario, there would be no yeast mitochondrial protein complexes left in SDS for dissociation by fluorinated alcohols.…”

“…Comparison to the results of two BN-SDS-PAGE studies on E. coli [12,13], revealed an overlap of only 9 integral membrane proteins and 11 peripheral proteins, indicating that, particularly for integral membrane proteins, the two techniques are complementary. For two of the proteins identified in the SDS-TFE-SDS-PAGE study, the preprotein translocase SecA and the mechanosensitive channel MscS, the oligomeric state and, in the latter case, its dissociation by TFE was confirmed in one dimensional SDS-PAGE gels using immunodetection [8,14].…”

“…This was achieved by replacing SDS with LDS, which does not precipitate at low temperatures, thus allowing the sample preparation and electrophoresis to be performed on ice. This method has been successfully applied to study the effect of TFE on the dissociation of E. coli MscS (mechanosensitive channel of small conductance), a heptameric complex of identical subunits that was stable in LDS-PAGE on ice, and could be dissociated by adding TFE [8]. When yeast mitochondria were separated by LDS-PAGE on ice and the resulting gel lanes were incubated with or without TFE, no additional off-diagonal spots were detected in the second dimension gel (compare Figs.…”

“…SDS-stable complexes were shown to dissociate under the influence of small, fluorinated, alcohols such as 2,2,2-trifluoroethanol (TFE) that are thought to disturb the local interactions between hydrophobic protein segments, and/or between the hydrophobic protein segments and the surrounding membrane lipids or SDS-molecules [5][6][7][8]. In addition, TFE has been used at high concentrations (≥50%) to improve the separation of membrane proteins from E. coli [9] and Streptococcus mutans [10] in the classic 2D iso-electric focusing (IEF)-SDS-PAGE approach, illustrating its potential to solubilize membrane proteins.…”

“…In addition, TFE has been used at high concentrations (≥50%) to improve the separation of membrane proteins from E. coli [9] and Streptococcus mutans [10] in the classic 2D iso-electric focusing (IEF)-SDS-PAGE approach, illustrating its potential to solubilize membrane proteins. In contrast, TFE is generally regarded as a stabilizing agent for soluble, globular proteins [8,11].…”