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 energygenerating 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.

Computational and Spectroscopic Characterization of the Photocycle of an Artificial Rhodopsin

“…The observation that while a PSB gives a 13- cis photoproduct, the rUSB gives rise to photoisomerization of the C15N bond is consistent with a recent study of a rhodopsin mimic variant of M2 . In this study, it was shown that an all - trans retinylidene can be photoisomerized to a stable 13- cis ,15- cis product; but only in its PSB state.…”

Human Cellular Retinol Binding Protein II Forms a Domain‐Swapped Trimer Representing a Novel Fold and a New Template for Protein Engineering

“…We next investigated the potential of the DS trimer as a new protein design template by engineering a metal binding site as a proof of principle. The engineering of a metal‐binding site to control oligomerization and create new function has been previously investigated by several research groups including us [20,36,42–44] . Herein, we wanted to show how the structural differences between monomer, dimer, and trimer can be used to create a metal‐binding site by minimal changes into the protein sequence.…”

“…The engineering of a metal-binding site to control oligomerization and create new function has been previously investigated by several research groups including us. [20,36,[42][43][44] Herein, we wanted to show how the structural differences between monomer, dimer, and trimer can be used to create a metal-binding site by minimal changes into the protein sequence. A characteristic feature of the DS trimer is the pseudo C 3 symmetry axis running through a volume of buried surface unique to the trimer structure ( Figure S4a).…”

“…Human cellular retinol binding protein II (hCRBPII) chaperones both retinol and retinal within the cell. Our group has engineered these templates to create rhodopsin mimics used to understand protein-based absorption tuning, protein-directed photoisomerization pathways, [35][36][37] new classes of fluorescent and photoswitchable fluorescent protein tags, [38] and pH sensors. [39,40] We recently reported that some variants of hCRBPII give rise to domain-swapped (DS) dimers.…”

Excited-state dynamics of the all-trans protonated retinal Schiff base in CRABPII-based rhodopsin mimics

“…On the other hand, the designable proteins are relatively stable on limited mutations, allowing us to study the photoreaction mechanisms through systematical mutagenesis. Geiger, Borhan et al .have been working on the artificial rhodopsin mimics for a long history and have provided several mimics with the human proteins CRABPII or the cellular retinol binding protein II (CRBPII) as scaffoldings (10; 11; 12; 13). The crucial component for the photoreaction, the retinal Schiff base (RSB), is correctly generated in these mimics between the chromophore retinal and the sidechain of the residue LYS in the scaffolding protein.…”