Kang, Bok Eum Leong, Lee Min Kim, Yoonkyung Miyazaki, Kenichi Ross, William N. Baker, Bradley J. 2024-01-19T14:33:36Z 2024-01-19T14:33:36Z 2021-10-21 2021-05-18 0006-3495 https://pubs.kist.re.kr/handle/201004/116985 The genetically encoded voltage indicators ArcLight and its derivatives mediate voltage-dependent optical signals by intermolecular, electrostatic interactions between neighboring fluorescent proteins (FPs). 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. One mutation to the FP (E222H) showed a voltage-dependent increase in fluorescence when excited at 390 nm, indicating the ability to affect the proton wire from the protonated chromophore to the H222 position. ArcLight-derived sensors 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 affect those pathways via conformational changes mediated by manipulating membrane potential. English CELL PRESS GREEN FLUORESCENT PROTEIN VOLTAGE-SENSITIVITY DYNAMICS PH TRANSMISSION INDICATOR NEURONS EVENTS Mechanism of ArcLight derived GEVIs involves electrostatic interactions that can affect proton wires Article 10.1016/j.bpj.2021.03.009 1 BIOPHYSICAL JOURNAL, v.120, no.10, pp.1916 - 1926 BIOPHYSICAL JOURNAL 120 10 1916 1926 scie scopus 000652829900007 2-s2.0-85103387602 Biophysics Biophysics Article GREEN FLUORESCENT PROTEIN VOLTAGE-SENSITIVITY DYNAMICS PH TRANSMISSION INDICATOR NEURONS EVENTS