Figure 5.

Effect of labeling MTSET before reconstitution. MscL was labeled with MTSET either before or after reconstitution into lipid vesicles. (a) DOPC vesicles were reconstituted with unlabeled MscL. Addition of 1 mM MTSET after 50 s had little effect, so that addition of 200 μM C₁₂E₈ after 400 s resulted in a large release of calcein. (b) MscL was labeled with 1 mM MTSET in OG micelles prior to reconstitution and then reconstituted with DOPC. As shown by the large release of calcein observed on addition of C₁₂E₈ after 350 s, the vesicles retained a large concentration of calcein. The experiments were repeated for vesicles containing a 1:1 molar ratio of DOPG:DOPC (c, d). When unlabeled MscL was reconstituted into a 1:1 mixture of DOPG:DOPC, addition of MTSET after 50 s resulted in a large release of calcein, so that subsequent addition of C₁₂E₈ resulted in only a small release (c). When labeled MscL was reconstituted into vesicles containing a 1:1 molar ratio of DOPG:DOPC, the vesicles retained only a small amount of calcein, as shown by the small response on addition of C₁₂E₈ after 100 s (d).