The crowding effects of 5% Ficoll 400 on WT and G168D MscS opening and closing. (A) Under symmetric ramps of saturating pressure, both WT and G168D MscS open at similar tensions yet G168D closes much more slowly, resulting in greater hysteresis of the closing portion of the activation curve. Upon the addition of 5% Ficoll, activation curves for both WT and G168D shift ∼10% to the right (shown in red). (B and C) Pulse-step protocols revealing the kinetics of channel closure (k_off) at different tensions for both WT and G168D. The current relaxation traces are shown before (B) and after (C) the addition of 5% Ficoll. Arrows indicate the pressure that was applied for the 2-s step during which the closing kinetics were monitored. (D) The closing rates from each of these curves (before and after 5% Ficoll) are plotted against the applied tension (n = 3 for each). The alteration of closing kinetics after 5% Ficoll is more pronounced for WT than G168D MscS. The inset shows a theoretical single barrier energy profile. The solid line represents the transition from closed to open in WT MscS, whereas the dashed line corresponds to G168D; the deeper and wider energy well for the open state of the mutant indicates a stabilization of the open state and a slower closing rate.