Engineered Functional Recovery of Microbial Rhodopsin without Retinal‐Binding Lysine

: Definition of rhodopsin is the retinal-binding membrane protein with the Schiff base linkage at a lysine on the 7th transmembrane helix. However, ~ 600 microbial rhodopsins lack retinal-binding lysine at the corresponding position (Rh-noK) among ~ 5500 known microbial rhodopsins, suggesting that Rh-noK has each functional role without chromophore. Here, we report successful functional recovery of Rh-noK. Two Rh-noKs from bacteria were heterologously expressed in Escherichia coli, which exhibited no color. When retinal-binding lysine was introduced, one of them gained visible color. Additional mutation of the Schiff base counterion further gained proton-pumping activity. Successful engineered functional recovery such as visible color and proton-pump activity suggests that the Rh-noK protein forms a characteristic structure of microbial rhodopsins.