Red-shifting mutation of light-driven sodium-pump rhodopsin

Abstract

Microbial rhodopsins are photoreceptive membrane proteins that transport various ions using light energy. While they are widely used in optogenetics to optically control neuronal activity, rhodopsins that function with longer-wavelength light are highly demanded because of their low phototoxicity and high tissue penetration. Here, we achieve a 40-nm red-shift in the absorption wavelength of a sodium-pump rhodopsin (KR2) by altering dipole moment of residues around the retinal chromophore (KR2 P219T/S254A) without impairing its ion-transport activity. Structural differences in the chromophore of the red-shifted protein from that of the wildtype are observed by Fourier transform infrared spectroscopy. QM/MM models generated with an automated protocol show that the changes in the electrostatic interaction between protein and chromophore induced by the amino-acid replacements, lowered the energy gap between the ground and the first electronically excited state. Based on these insights, a natural sodium pump with red-shifted absorption is identified from Jannaschia seosinensis .

Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping

“…A similar red-shift by the mutation of an -OH bearing residue at the same position was reported in several type-1 rhodopsins 21,22 , and SzR4 T184 has a similar effect on the excitation energy of the retinal π-electron.…”

“…SzR4 P158 in TM5 and T187 in TM7 are highly conserved in 96% and 100% of the SzRs (Fig. 4b), and the homologous residues in type-1 rhodopsins play a color tuning role 21,22 . Mutating the former to threonine or the latter to alanine makes λmax longer for many type-1 rhodopsins.…”

Exploration of natural red-shifted rhodopsins using a machine learning-based Bayesian experimental design

“…The differences in amino acids in three of 24 retinal-surrounding residues are known to play a color-tuning role in natural rhodopsins without affecting their biological function. These correspond to positions 93, 186, and 215 in BR (BR Leu93, Pro186, and Ala215, respectively) 16 . Position 93 is known to be diversified in the PR family (the well-known position 105 in PRs).…”

“…Position 215 in BR is also known to have a colour-tuning role. The mutation from alanine to threonine or serine (A/TS switch) has a blue-shifting effect of 9–20 nm 16,34–36 . Five of seven genes that showed blue-shifted λ max compared with the base wavelengths have threonine or serine at this position, suggesting that these types of genes should be avoided to explore red-shifted rhodopsins.…”

“…The A-to-N mutation at this position had a smaller effect (4–7 nm) 20,35 than that of the A-to-S/T mutation; thus, the difference between alanine and asparagine is not so critical to explore red-shifted rhodopsins. Position 186 in BR is proline in most microbial rhodopsins (in 98.7% of the 3,064 candidate genes), and the mutation to non-proline amino acids induces red-shift of absorption 16 . We identified sodium pump rhodopsin (NaR) from Parvularcula oceani , which also has a threonine at this position, and showed 10-nm longer absorption than the base wavelength.…”