Mimicking Microbial Rhodopsin Isomerization in a Single Crystal

Abstract

Bacteriorhodopsin represents the simplest, and possibly most abundant, phototropic system requiring only a retinal-bound transmembrane protein to convert photons of light to an energy-generating proton gradient. The creation and interrogation of a microbial rhodopsin mimic, based on an orthogonal protein system, would illuminate the design elements required to generate new photoactive proteins with novel function. We describe a microbial rhodopsin mimic, created using a small soluble protein as a template, that specifically photoisomerizes all- trans to 13- cis retinal followed by thermal relaxation to the all- trans isomer, mimicking the bacteriorhodopsin photocycle, in a single crystal. The key element for selective isomerization is a tuned steric interaction between the chromophore and protein, similar to that seen in the microbial rhodopsins. It is further demonstrated that a single mutation converts the system to a protein photoswitch without chromophore photoisomerization or conformational change.

Figure 1.

All-trans retinylidene chromophore of bacteriorhodopsin photoisomerized to the 13-cis isomer followed by thermal regeneration of the all-trans isomer.

Figure 2.

(a) Photocycle of CRABPII hexamutant M1 (R111K:Y134F:T54V:R132Q:P39Y:R59Y) bound to retinal. The photoinduced isomerization of the imine functionality leads to changes in its pK_a and consequently its protonation state. Hydrophilic and hydrophobic environments of the imine nitrogen atom for the cis and trans geometry, respectively, are highlighted in the figure obtained from the crystal structure of each form (PDB IDs 4YFP and 4YFQ). (b) The present study aims to induce photoisomerization to the C13 isomer while maintaining the protonated iminium. (c) All-trans-retinal-bound M1-CRABPII, with key residues highlighted.

Figure 3.

(a) UV–vis absorption spectrum of all-trans-retinal bound M1-L121E in the dark state (black spectrum) and after green light irradiation (long-pass filter, >500 nm, green spectrum); blue light irradiation (440 ± 20 nm, blue spectrum), and UV irradiation (UV band-pass filter, 300–400 nm, cyan spectrum). (b) Hydrogen bonding network between the imine hydrogen of R111K and L121E, P39Y, H₂O-303, R132Q, and Ser12. (c) Overlay of M1 (blue carbons) with all-trans-retinal bound M1-L121E (green carbons). (d) Electron density (contoured at 1σ) of all-trans-retinylidene in M1-L121E in the dark state (green carbons) vs 13-cis-15-syn-retinal imine (magenta carbons) generated after 5 min of laser irradiation at 399 nm. (e) Overlay of all-trans-retinylidine-bound M1-L121E in the dark (green) with the 13-cis-15-syn imine (magenta) generated after 5 min of laser irradiation at 399 nm, showing the movement of Lys111 and Glu121 and the rotation of the β-ionone ring in 13-cis upon isomerization. (f) Overlay of all-trans-retinal bound M1-L121E imine (green) and all-trans-retinal bound bacteriorhodopsin (yellow carbons, PDB code 1C3W) illustrating how the P39YL121E interaction mimics Trp86 in bacteriorhodopsin relative to retinal. All heteroatoms are colored by type, with N being blue and O being red.

Figure 4.

(a) Overlay of the all-trans-retinal/M1-L121E complex in the dark (green) and after laser irradiation followed by incubation in the dark (25 min, orange) highlighting the structural similarity of the chromophore before and after a complete photocycle. (b) Overlay of the all-trans-retinal/M1-L121E complex in the dark (green) with the 13-cis-retinal/M1-L121E:A32Y complex. (c) Aromatic residues located in the binding pocket of 13-cis-bound M1-L121E:A32Y. (d) Bacteriorhodopsin (PDB code 1C3W) binding pocket where the chromophore is sandwiched with aromatic residues.

Figure 5.

(a) UV–vis spectra of M1-L121Q before and after irradiation with green light, showing a high/low pK_a system where green light irradiation leads to decreased PSB and increased SB absorption. (b) Overlay of the all-trans-retinal-bound M1-L121Q imine before (green carbons) and after (salmon carbons) laser irradiation at 532 nm, clearly showing the Gln121 movement. The all-trans retinal bound M1-L121Q structure features a direct hydrogen bond between the nitrogen atom of the imine and Gln121 (3.0 Å). Laser irradiation (532 nm) of crystals does not result in the isomerization of any bonds; however, L121Q swings away from the imine (now ~4.7 Å), leading to the low pK_a form of the imine and SB formation.