Abstract
Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here, we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths was 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 shorten and lengthen the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild-type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients toward 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.
Highlights
Anion channelrhodopsins (ACRs) are light-g ated anion channels that undergo photoisomerization at the retinal chromophore, which is covalently attached to a conserved lysine residue via the protonated Schiff base, from all-trans to 13-cis
It was proposed that Asp234 was protonated in the wild-type GtACR1 (Kim et al, 2018; Sineshchekov et al, 2016; Yi et al, 2016; Kandori, 2020) from the following results: (i) the absorption wavelength remains unchanged upon the D234N mutation (Kim et al, 2018; Sineshchekov et al, 2016; Yi et al, 2016); (ii) the C=C stretching frequency of the retinal is not significantly affected upon the D234N mutation in resonance Raman spectroscopy, which implies that the electrostatic interaction between the retinal and the protein environment remains unchanged (Yi et al, 2016); and (iii) the C=O stretching frequencies of 1740 (–)/1732 (+) cm–1 for a protonated carboxylate, which is observed in the wild-t ype GtACR1, disappear in the D234N GtACR1 at 77 K (Kim et al, 2018)
The present results show that Asp234 is deprotonated in the wild-t ype GtACR1, as indicated by the following findings. (i) The E68Q/D234N mutation leads to an increase in the absorption wavelength (Table 4), which indicates that Glu68 or Asp234 is deprotonated in the wild-type GtACR1 (Table 1). (ii) The absorption wavelength in the D234E GtACR1 is shorter than in the wild-type protein (Table 4), which can be explained only by the presence of a deprotonated acidic residue (Table 1, Supplementary file 1D). (iii) The calculated pKa value of –5 for Asp234 is lower than that of –2 for the corresponding residue Asp212 in BR (Saito et al, 2012; Table 2)
Summary
Anion channelrhodopsins (ACRs) are light-g ated anion channels that undergo photoisomerization at the retinal chromophore, which is covalently attached to a conserved lysine residue via the protonated Schiff base, from all-trans to 13-cis. Natural ACRs were identified in the cryptophyte Guillardia theta (GtACR1 and GtACR2) (Govorunova et al, 2015). Microbial rhodopsins have acidic residues or Cl– at the Schiff base moiety to stabilize the protonated Schiff base as counterions. Counterions play a major role in determining the absorption wavelength and the function of the protein (Tsujimura and Ishikita, 2020). The X-ray crystal structures of GtACR1 show that two acidic residues, Glu and Asp234, exist at the corresponding positions (Figure 1; Kim et al, 2018; Li et al, 2019)
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