Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.06.328245v1?rss=1 Authors: Kang, B. E., Leong, L. M., Kim, Y., Miyazaki, K., Ross, W., Baker, B. J. Abstract: The genetically encoded voltage indicators, ArcLight and its derivatives, mediate voltage dependent optical signals by intermolecular, electrostatic interactions between neighboring fluorescent proteins (FPs) via proton wires. A random mutagenesis event placed a negative charge on the exterior of the FP resulting in a greater than 10-fold improvement of the voltage-dependent optical signal. Repositioning this negative charge on the exterior of the FP reversed the polarity of voltage-dependent optical signals suggesting the presence of 'hot spots' capable of interacting with the negative charge on a neighboring FP thereby changing the fluorescent output. To explore the potential effect on the chromophore state, voltage-clamp fluorometry was performed with alternating excitation at 390 nm followed by excitation at 470 nm resulting in several mutants exhibiting voltage-dependent, ratiometric optical signals of opposing polarities. However, the kinetics, voltage ranges, and optimal FP fusion sites were different depending on the wavelength of excitation. These results suggest that the FP has external, electrostatic pathways capable of quenching fluorescence that are wavelength specific. ArcLight-derived GEVIs may therefore offer a novel way to map how conditions external to the {beta}-can structure can affect the fluorescence of the chromophore and transiently manipulate those pathways via conformational changes mediated by whole cell voltage clamp. Copy rights belong to original authors. Visit the link for more info