Abstract
Rho-GC is a rhodopsin-guanylyl cyclase gene fusion protein which is critical to zoospore phototaxis for the aquatic fungus Blastocladiella emersonii. This unique fusion has generated much speculation due to its potential as an optogenetic tool through the manipulation of cell signaling pathways involving cyclic nucleotides. To increase the utility of Rho-GC in optogenetic studies, two mutants with blue shifted absorption maxima (E254D, λmax = 390 nm; D380N, λmax = 506 nm) and one mutant with red shifted absorption maxima (D380E, λmax = 533 nm) relative to wild-type protein (λmax = 527 nm) have been designed. These spectral tuning mutants widen the λmax of the chromophore by over 40nm, allowing for selective activation using different wavelengths of light. These mutants have also identified residues involved in the complex counterion responsible for stabilizing retinal binding. More structural information is needed to engineer larger spectral shifts; to that end, crystal trials have been conducted but have yet to yield any results. Regardless, the spectral tuning mutants have yielded important structural information while expanding the utility of this optogenetic tool.