Abstract
RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus Blastocladiella emersonii It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution. The protein exhibits robust light-dependent guanylyl cyclase activity, whereas a truncated form lacking the 17- to 20-kDa N-terminal domain is completely inactive under identical conditions. Moreover, we designed several RhoGC mutants to increase the utility of the protein for optogenetic studies. The first class we generated has altered absorption spectra designed for selective activation by different wavelengths of light. Two mutants were created with blue-shifted (E254D, λmax = 390 nm; D380N, λmax = 506 nm) and one with red-shifted (D380E, λmax = 533 nm) absorption maxima relative to the wild-type protein (λmax = 527 nm). We also engineered a double mutant, E497K/C566D, that changes the enzyme to a specific, light-stimulated adenylyl cyclase that catalyzes the formation of cAMP from ATP. We anticipate that this expression/purification system and these RhoGC mutants will facilitate mechanistic and structural exploration of this important enzyme.
Highlights
RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus Blastocladiella emersonii
We present two groups of mutants designed to expand the utility of the protein in optogenetic studies: a group of spectral tuning mutants that hypso- and bathochromically shift the visible absorption maximum of the protein over a range of about 40 nm and a substrate specificity mutant that changes the protein from a guanylyl cyclase into an adenylyl cyclase
The gene for the protein was expressed to high levels in HEK293-GnT1Ϫ cells in culture, and the protein was purified to near homogeneity, free of proteolytic contaminants, by a tandem immunoaffinity chromatography protocol that used two different epitope tags: C8 [8] at the N terminus and 1D4 [9, 10] at the C terminus of the protein
Summary
Tions has been expanded by the identification of a very unusual protein from the aquatic fungus Blastocladiella emersonii [5]. The phototactic signaling pathway in B. emersonii shares similarities with the cyclic nucleotide pathway of vertebrate vision despite the fact that the rhodopsin domain of RhoGC is distantly related to the type II rhodopsins [5]. The 2014 report by Avelar et al [5] was followed quickly by two publications demonstrating the exciting potential of RhoGC to expand the tools available for optogenetic studies, for controlling cyclic nucleotide signaling pathways [6, 7]. We present two groups of mutants designed to expand the utility of the protein in optogenetic studies: a group of spectral tuning mutants that hypso- and bathochromically shift the visible absorption maximum of the protein over a range of about 40 nm and a substrate specificity mutant that changes the protein from a guanylyl cyclase into an adenylyl cyclase
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