Proton-mediated gating mechanism of anion channelrhodopsin-1

ABSTRACT Anion channelrhodopsin from Guillardia theta ( Gt ACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here we investigate mutant Gt ACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths were also analyzed, using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shortens and lengthens the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild type Gt ACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients towards the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.