The effects of chemical modification of MscL have been followed under isoosmotic conditions in a straightforward liposomal efflux assay, by using fluorescence changes upon the release of a reporter (see Supporting Information). Briefly, chemically modified channels were incorporated in liposomes consisting of synthetic lipids by a detergentmediated reconstitution method [24] in the presence of calcein, a self-quenching fluorescent dye. After chromatographic removal of external dye, the resulting proteoliposomes (average size 200nm, as determined by dynamic light scattering) were analyzed for channel activity at different pH values. Channel opening led to the release of the dye from the liposomal interior, and the resulting concentration decrease and dequenching upon entering the surrounding buffer could be monitored as an increase in fluorescence. Controls with liposomes lacking MscL-G22C, or containing unmodified MscL-G22C or wild-type MscL (with the C-terminal His tag that is present in all proteins described here), did not show any release activity (see Supporting Information). pH-induced release through MscL modified with 1 could not be observed by the assay described above between pH 6 and 8, the pH limits of the assay. To access lower pH ranges, pyridine-modified channels were analyzed at the singlemolecule level by measuring the ionic current flowing through the channel in response to the pH in patch-clamp experiments. Spontaneous channel opening was observed at pH 5.2, but at higher pH values the channels still required tension to open (see Supporting Information). Modulator 2 was designed to have a higher pKa value, within the physiological range as well as the pH interval of the efflux assay. Channels modified with 2 did indeed show pH-dependent activity in efflux assays (Figure 2 a), a result confirmed with patch-clamp measurements (Figure 2b and c). Thus, we showed for the first time that it is possible to open the channel in response to pH in synthetic lipids without applying any tension, in both patch-clamp and liposomal efflux setups. It is known that the gating behavior of MscL channels can be correlated with the hydrophilicity of the residues present in the pore constriction.[10] From this point of view, the pyridine derivatives are not ideal modulators as they are quite hydrophobic. To address this issue and improve the liposomal efflux efficiency, pH modulators based on a different structural motif (3–5) were designed. We aimed specifically at increased hydrophilicity, a pKa value tunable in the range pH6–8, synthetic availability, and specific and efficient coupling to the protein.

Proteoliposomes containing channels modified with modulators 3–5 responded to pH and released more calcein at pH values below the pKa of the modulator used. In contrast, control liposomes without MscL were stable at all pH values analyzed (Figure 3). The pH interval in which the channel is activated is dependent on the pKa and hydrophobicity of the modulator. When the hydrophobicity of the pH modulator was higher as a result of the additional methyl groups, the modified channel was harder to open at higher pH values, where the modulator is mainly in its uncharged form. A particularly interesting case is the channel modified with 5 (Figure 3 c), which has the highest hydrophobicity of the three modulators as well as the lowest pKa. At pH7. 4, the