Red Shift of the Absorption Maximum of Mutant Exiguobacterium sibiricum Proteorhodopsin: Influence of Amino Acid Substitutions in Helices E and F

Abstract Objective: To engineer a mutant variant of the Exiguobacterium sibiricum proteorhodopsin (ESR) by introducing amino acid substitutions in helices E and F, based on the carotenoid-binding site of xanthorhodopsin (XR) from Salinibacter ruber, and to investigate the effects of these mutations on the absorption spectrum, photocycle kinetics, and carotenoid-binding ability. Methods: Three mutant variants were constructed: Car3 (substitutions Y147G, C151T, W154F in helix E), Car4 (substitution of fragment A199-L202 in helix F with the corresponding XR fragment M208-M211), and Car5 (combining both modifications). Genes were obtained by SOE-PCR and expressed in E. coli. Proteins were purified by metal-affinity chromatography. Absorption spectra were recorded at pH 5–9, and photocycle kinetics were measured at 410, 510, 550, and 590 nm using laser flash photolysis. Carotenoid binding was tested by co-expression of the rhodopsin genes with carotenoid biosynthesis genes. Results and Discussion: Car5 exhibited a red shift of 9–16 nm compared to wild-type ESR across pH 5–9 (e.g., 542 nm at pH 7), while Car3 and Car4 showed no substantial shift. The pH dependence of Car5's absorption maximum revealed two transitions: a red shift from 538 to 550 nm (pKa 3.9) and a blue shift to 535 nm (pKa 6.8). This contrasts with wild-type ESR, which exhibits only blue shifts (pKa 6.0 and 9.1) and no red shift in this pH range. In the photocycle, Car5 formed the M intermediate already at pH 8 (unlike wild type), with faster M formation and decay at pH 9. Car5 also lacked the slow component of M formation observed in Car3, Car4, and wild type. No carotenoid binding was detected for Car3 or Car5, suggesting that additional mutations are required. These changes likely arise from altered electrostatic interactions and protonation states of the Schiff base counterion (D85 and H57), as well as conformational adjustments near the retinal β-ionone ring. Conclusions: Combined mutations in helices E and F of ESR produce a significant red shift in the absorption maximum and alter photocycle kinetics, shifting M intermediate formation to lower pH. These findings contribute to the rational design of microbial rhodopsins with tailored spectral properties for optogenetic applications. However, creation of a functional carotenoid-binding site in ESR requires further mutagenesis.