Engineering inducible dipoles to develop better fluorescent biosensors

Abstract

ArcLight is a genetically encoded voltage indicator (GEVI) utilizing a pH-sensitive green fluorescent protein, super ecliptic pHluorin (SE). SE can optically report changes in pH but requires the additional A227D mutation to generate a large voltage-dependent optical signal. There must exist than electrodynamic pathways from the protein’s surface that affect the internal chromophore. We investigated the impact of external amino acids flanking an internal amino acid near the chromophore. A novel ArcLight-derived GEVI with the internal F165T mutation resulted in a multicomponent optical signal transition when the membrane potential was altered. Introducing asymmetric polarity to the flanking external amino acids of F165T (N164F/K166T) results in a similar multicomponent response but with lower dynamic range. However, inverting the direction of the external polarity (N164T/K166F) eliminated the secondary component and increased the dynamic range. Alphafold predictions suggest that the orientation of the F165T side chain differs depending on the external chemistry. We therefore altered the degree of the polarity by maintaining 166F and mutating 164 to A/S/E/D/H/K/R/Y. 164E/166F yielded the largest signal. Surprisingly, 164D/166F yielded the worst signal. The external polarity was then reversed (164F/166X), where X represents ？D/E/K/R. Inversion of the external polarity affected the voltage-dependent signal suggesting that the orientation of internal side chains of the β-can structure can be influenced by external amino acids. These observations allows us to map how external charge conditions can influence the photo-physical properties of the protein, paving the way for advancements in biosensor development. This research is funded by KIST (2E32162).