Redesign of optogenetic actuators and characterization of the extended photocycle mutant L348F.
A, optimized versions of OptoBAX created for reduction of light-independent cell death and extension of the photocycle. Western blot (α-mCherry, inset) lanes: 1, Cry2.531.mCherry; 2, Cry2.531.L348F.mCherry; 3, Cry2.531.mCherry.BAX; 4, Cry2.531.L348F.mCherry.BAX; 5, Cry2PHR.mCherry.BAX. B, demonstration of the elongated photocycle in the Cry2 L348F mutant. Images shown are before and 5 and 10 min after activation with a 150-ms pulse of 470-nm light. C, plot of normalized fluorescence data from the experiment shown in B. Error bars represent standard deviations from six replicate measurements of light-initiated recruitment using the method described by Haar et al. (53). Scale bar = 10 μm.