Abstract
Several genetically encoded fluorescent sensors of voltage were created by systematically truncating the length of the linker sequence between the voltage-sensing domain and the position of the fluorescent protein, Super Ecliptic A227D. In addition to varying the length, the amino acid composition at the fusion site for the fluorescent protein was modified. Both linker length and amino acid composition affected the size and voltage sensitivity of the optical signal. The truncation mutants revealed a potential structural periodicity with a maximum signal three amino acids from the voltage-sensing domain and another maximum 11 amino acids from the voltage-sensing domain. These results confirm that the linker length and composition can fine tune the size and voltage range of the sensor. The potential periodicity suggests that the orientation of the fluorescent protein could be important for improving the signal size implicating dimerization of the fluorescent protein.
Highlights
In order to faithfully represent neuronal activity, a genetically encoded fluorescent voltage sensor (GeFVS) must respond to changes in membrane potential with a rapid and large fluorescent change
ArcLight, a GeFVS with one of the largest optical signals reported, yields a 30 to 40% ΔF∕F∕100 mV depolarization of the plasma membrane.[1,2]. This large signal is due to the combination of a mutation to the fluorescent protein (FP), Super Ecliptic pHlorin A227D (SE 227D) and the optimization of the linker length between the voltage-sensing domain (VSD) and the FP
The amino acid composition at the fusion site of the FP affected the fluorescent response. These results suggest that the orientation of the FP is important for the voltage-dependent optical signal
Summary
In order to faithfully represent neuronal activity, a genetically encoded fluorescent voltage sensor (GeFVS) must respond to changes in membrane potential with a rapid and large fluorescent change. A large fluorescent change is needed since the total amount of responsive probe is limited to the plasma membrane. The brightest GeFVS consists of a transmembrane, voltage-sensing domain (VSD) coupled to a cytoplasmic, fluorescent protein (FP) via a linker sequence. ArcLight, a GeFVS with one of the largest optical signals reported, yields a 30 to 40% ΔF∕F∕100 mV depolarization of the plasma membrane.[1,2] This large signal is due to the combination of a mutation to the FP, Super Ecliptic pHlorin A227D (SE 227D) and the optimization of the linker length between the VSD and the FP. ArcLight uses the VSD from the Ciona voltage-sensing phosphatase (VSP) gene. Some of the fastest GeFVS published utilize the VSD of the zebrafish VSP (Ref. 3) or the VSD from the
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