Directed evolution of the genetically encoded zinc(II) FRET sensor ZapCY1

Abstract

Zinc(II) ions (Zn²⁺) play an essential role in living systems, with their delicate concentration balance differing among the various intracellular organelles. The spatiotemporal distribution and homeostasis of Zn²⁺ can be monitored through photoluminescence imaging using zinc sensors. Among such biosensors, genetically encoded fluorescent sensor proteins are attractive tools owing to their subcellular localization advantage and high biocompatibility. However, the limited fluorescent properties of these proteins, such as their insufficient quantum yield and dynamic range, restrict their practical use. In this study, we developed an expression-screening-directed evolution system and used it to improve ZapCY1, a genetically encoded fluorescence resonance energy transfer (FRET) sensor. After four rounds of directed evolution, the FRET dynamic range of the modified sensor (designated ZapTV-EH) was increased by 1.5-1.7-fold. With its enhanced signal-to-noise ratio and ability to detect a wide Zn²⁺ concentration range, ZapTV-EH proves to be a better visualization tool for monitoring Zn²⁺ at the subcellular level. Combined with the simplified subcloning and expression steps and sufficient mutant libraries, this directed evolution system may provide a more simple and efficient way to develop and optimize genetically encoded FRET sensors through high-throughput screening.

Keywords: Directed evolution; FRET; Genetically encoded fluorescent sensor; Zinc sensor.