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• W3137489265 startingPage "1916" @default.
• W3137489265 abstract "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 β-can structure can affect the fluorescence of the chromophore and transiently affect those pathways via conformational changes mediated by manipulating membrane potential." @default.
• W3137489265 created "2021-03-29" @default.
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• W3137489265 date "2021-05-01" @default.
• W3137489265 modified "2023-10-16" @default.
• W3137489265 title "Mechanism of ArcLight derived GEVIs involves electrostatic interactions that can affect proton wires" @default.
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• W3137489265 doi "https://doi.org/10.1016/j.bpj.2021.03.009" @default.
• W3137489265 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8204334" @default.
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