Figure 6

Thermostability and functionality of MelB_St solubilized in DDM or individual novel agents (TMG-As: TMG-A11, TMG-A12, TMG-A13 and TMG-A14; TMG-Ts: TMG-T11, TMG-T12, TMG-T13 and TMG-T14). E. coli membranes containing MelB_St were mixed with the indicated detergent, and then kept at 0 °C or an elevated temperature (45, 55, or 65 °C) for 90 minutes. (a) Western blott analysis. The amount of soluble MelB_St after ultracentrifugation was detected by penta-His-HRP antibody. The protein samples were initially separated on SDS-15%PAGE gels. (b) Histogram. The density representing the soluble MelB_St in individual detergents detected in panel (a) was measured by ImageQuant software and expressed as a percentage (%) relative to that present in the untreated membrane sample (b). Error bars, SEM, n = 3. (c) MelB Trp→ D²G FRET reversal functional asssay. Sample preparations and FRET measurements are described in the Methods. The FRET signals were monitored over time. D²G at 10 μM was added at the 1-min time point and melibiose (black trace) at a saturating concentration added at the 2-min time point. Control experiments were carried out by adding water (gray trace) instead of melibiose at the 2-min time point. (d) Relative values for FRET reversal were obtained by calculating fluorescent intensity decrease (at 2-min point)/increase (at 1-min point).