Protonation of Asp116 and distortion of the all-trans retinal chromophore in Krokinobacter eikastus rhodopsin 2 causes a redshift in absorption maximum upon dehydration.

Water is usually indispensable for protein function. For ion-pumping rhodopsins, water molecules inside the proteins play an important role in ion transportation. In addition to amino acid residues, water molecules regulate the colors of retinal proteins.

Covalent Bond between the Lys-255 Residue and the Main Chain Is Responsible for Stable Retinal Chromophore Binding and Sodium-Pumping Activity of Rhodopsin 2.

Microbial rhodopsins are light-receptive proteins with various functions triggered by the photoisomerization of the retinal chromophore from the all- to 13- configuration. A retinal chromophore is covalently bound to a lysine residue in the middle of the seventh transmembrane helix via a protonated Schiff base. Bacteriorhodopsin (BR) variants lacking a covalent bond between the side chain of Lys-216 and the main chain formed purple pigments and exhibited a proton-pumping function.

Inverse Hydrogen-Bonding Change Between the Protonated Retinal Schiff Base and Water Molecules upon Photoisomerization in Heliorhodopsin 48C12.

Heliorhodopsin (HeR) is a new class of the rhodopsin family discovered in 2018 through functional metagenomic analysis (named 48C12). Similar to typical microbial rhodopsins, HeR possesses seven transmembrane (TM) α-helices and an all--retinal covalently bonded to the lysine residue on TM7 via a protonated Schiff base. Remarkably, the HeR membrane topology is inverted compared with that of typical microbial rhodopsins.

Mechanism of Inward Proton Transport in an Antarctic Microbial Rhodopsin.

Although the outward-directed proton transport across biological membranes is well studied and its importance for bioenergetics is clearly understood, inward-directed light-driven proton pumping by microbial rhodopsins has remained a mystery both physiologically and mechanistically. A new family of Antarctic rhodopsins, which is a subgroup within a novel class of schizorhodopsins reported recently, includes a member, denoted as AntR, which proved amenable to extensive characterization with experiments and computation. Phylogenetic analyses identify AntR as distinct from the well-studied microbial rhodopsins that function as outward-directed ion pumps, and bioinformatics sequence analyses reveal amino acid substitutions at conserved sites essential for outward proton pumping.

Schizorhodopsins: A family of rhodopsins from Asgard archaea that function as light-driven inward H pumps.

Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H pump.

Infrared spectroscopic analysis on structural changes around the protonated Schiff base upon retinal isomerization in light-driven sodium pump KR2.

Krokinobacter rhodopsin 2 (KR2) was discovered as the first light-driven sodium pumping rhodopsin (NaR) in 2013, which contains unique amino acid residues on C-helix (N112, D116, and Q123), referred to as an NDQ motif. Based on the recent X-ray crystal structures of KR2, the sodium transport pathway has been investigated by various methods. However, due to complicated structural information around the protonated Schiff base (PRSB) region in the dark state and lack of structural information in the intermediates with sodium bound in KR2, detailed sodium pump mechanism is still unclear.

Crystal structure of heliorhodopsin.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics. They are widely present in bacteria, archaea, algae and algal viruses. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins.

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

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.

A distinct abundant group of microbial rhodopsins discovered using functional metagenomics.

Many organisms capture or sense sunlight using rhodopsin pigments, which are integral membrane proteins that bind retinal chromophores. Rhodopsins comprise two distinct protein families , type-1 (microbial rhodopsins) and type-2 (animal rhodopsins). The two families share similar topologies and contain seven transmembrane helices that form a pocket in which retinal is linked covalently as a protonated Schiff base to a lysine at the seventh transmembrane helix.

Hydrogen-bonding network at the cytoplasmic region of a light-driven sodium pump rhodopsin KR2.

Light-driven sodium-pumping rhodopsins are able to actively transport sodium ions. Structure/function studies of Krokinobacter eikastus rhodopsin 2 (KR2) identified N61 and G263 at the cytoplasmic surface constituting the "Ion-selectivity filter" for sodium ions, while retinal Schiff base acts as the light "Switch and Gate" for transport of sodium ions. Q123 is located between the two regions, and plays an important role for the pump function, which was implicated by functional, spectroscopic, X-ray crystallographic and computational studies.

Long-distance perturbation on Schiff base-counterion interactions by His30 and the extracellular Na-binding site in Krokinobacter rhodopsin 2.

Krokinobacter rhodopsin 2 (KR2), a light-driven Na+ pump, is a dual-functional protein, pumping protons in the absence of Na+ when K+ or larger alkali metal ions are present. A specific mutation in helix A near the extracellular Na+ binding site, H30A, eliminates its proton pumping ability. We induced structural changes in H30A by altering the alkali metal ion bound at the extracellular binding site, and observed a strong electrostatic interaction between the Schiff base and counterion and torsion around the Schiff base as revealed by solid-state nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopies.