Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties

Abstract

Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for lightharvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal binding pocket of Gloeobacter violacaeus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to +/-80 nm, extending the protein's light absorption significantly beyond the range of known natural PPRs. While proton pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurrred blue and red shifts of 42 nm and 22 nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while redshifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty-four of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the wild-type protein.

Directed evolution of a far-red fluorescent rhodopsin

“…We believe improvements will be possible upon further directed evolution. Finally, the absorption maxima of fluorescent Arch variants reported here are significantly red-shifted compared with that of the wild-type protein (as are previously reported fluorescent GR variants) (15). Although the exact nature between the degree of red shift and fluorescence is not clear at this point, it would be interesting to investigate the effects on fluorescence of unnatural retinal analogs, which have been used previously to red-shift the λ max of bacteriorhodopsin by hundreds of nanometers (32).…”

“…S1). The mutations of a bright GR variant discovered in our previous directed evolution study of GR (15), GR(D121E/ T125C/A256M), which contains the desirable D→E substitution at the Schiff-base counter ion, map to D95E, T99C, and A225M in Arch, respectively (Fig. S1).…”

“…An alternative approach is utilization of mutations of the Schiff-base counter ion (15) to increase the fluorescence yield in the initial (unphotolyzed) state. Although further study will be required to establish the biophysical role of specific mutations in improving Arch fluorescence, we expect that further improvements to fluorescence can be found by continued optimization of the retinal binding pocket.…”

“…Site-saturation libraries were generated using a modified version of the QuikChange method (Stratagene), as previously described (15). For the single-site-saturation libraries, a mix of three mutagenic primers containing the triplet sequences NDT, VHG, and TGG were used in ratio of 12:9:1 for mutagenic PCR amplification (Table S1, primers [7][8][9][10][11][12][13][14][15][16][17][18][19][20]. For the double-site-saturation library, a mixture of 9 mutagenic primers (and a reverse primer) was used for PCR amplification (Table S1, primers [21][22][23][24][25][26][27][28][29][30].…”

“…We recently used directed evolution to tune the absorption maximum (λ max ) of Gloeobacter violaceus rhodopsin (GR), shifting it by ±80 nm (15). We found that a subset of red-shifted GR variants exhibited increased levels of fluorescence in the farred, demonstrating that rhodopsin fluorescence could be increased through mutations in the protein retinal-binding pocket to a greater extent than observed earlier for variants with protonated Schiff-base counter ion (16,17).…”