External Charges Influence Fluorescent Protein Proton Wires

Abstract

Genetically encoded voltage indicators (GEVIs) based on the fluorescent protein Superecliptic pHluorin have been shown to alter fluorescent output in a voltage-dependent manner. The dimer affinity of the fluorescent proteins (FP) and specific charged residues in the dimer interface are involved in the mechanism of how the fluorescent output changes have been implicated in the optical signal. Here we show that the critical negative charge on the exterior of the β-can structure of the FP (A227D) can affect proton wires that ultimately affect the chromophore of the FP. When the negative charge is moved along the exterior of the β-barrel, the polarity of the voltage-dependent optical signal inverts. One hypothesis to explain this behavior is that the transient movement of the negative charge shifts the equilibrium of the protonation state of the chromophore. To test this hypothesis, alternating the excitation wavelength from 390 nm to 470 nm was performed revealing that the kinetics of the optical signal, voltage range of the optical signal, and the optimal linker length were wavelength dependent. These results suggest that the proton wires capable of responding to the external negative charge of a neighboring probe are different for the protonated chromophore and the anionic chromophore. To further investigate the effect on proton wires, several E222 mutations were tested which has been implicated as the proton release point for excited state proton transfer of GFP. The results imply that ArcLight-derived GEVIs can be used to alter and thereby study proton wires of fluorescent proteins. Combined with structural data, these GEVIs may enable the mapping of proton wires, which would enable the development of voltage sensors with large dynamic range.