Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.

Silapetere, A., Hwang, S., Hontani, Y., Fernandez Lahorem, R. G., Balke, J., Velazquez Escobar, F., Tros, M., Konold, P. E., Matis, R., Croce, R., Walla, P. J., Hildebrandt, P., Alexiev, U., Kennis, J. T. M., Sun, H., Utesch, T., and Hegemann, P. (2022). QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins. Nat Commun, doi: 10.1038/s41467-022-33084-4.