X-ray Crystallographic Structure and Oligomerization of Gloeobacter Rhodopsin

Abstract

Gloeobacter rhodopsin (GR) is a cyanobacterial proton pump which can be potentially applied to optogenetics. We solved the crystal structure of GR and found that it has overall similarity to the homologous proton pump from Salinibacter ruber , xanthorhodopsin (XR). We identified distinct structural characteristics of GR’s hydrogen bonding network in the transmembrane domain as well as the displacement of extracellular sides of the transmembrane helices relative to those of XR. Employing Raman spectroscopy and flash-photolysis, we found that GR in the crystals exists in a state which displays retinal conformation and photochemical cycle similar to the functional form observed in lipids. Based on the crystal structure of GR, we selected a site for spin labeling to determine GR’s oligomerization state using double electron–electron resonance (DEER) spectroscopy and demonstrated the pH-dependent pentamer formation of GR. Determination of the structure of GR as well as its pentamerizing propensity enabled us to reveal the role of structural motifs (extended helices, 3-omega motif and flipped B-C loop) commonly found among light-driven bacterial pumps in oligomer formation. Here we propose a new concept to classify these pumps based on the relationship between their oligomerization propensities and these structural determinants.

The chirality origin of retinal-carotenoid complex in gloeobacter rhodopsin: a temperature-dependent excitonic coupling

“…In summary, it can be concluded that the CD spectrum of gR originated from the excitonic coupling between retinal chromophores located in a protein pentameric form (Fig. 8b) 15,33 . Due to change of temperature, the His87-Asp121 pair arrangement is altered leading to pentamer to monomer transition and the corresponding change of CD spectra at pH 5 and vanishing of CD exciton 9 .…”

“…The CD spectra of native gR as well as in the presence of different types of carotenoids were studied previously, and it was proposed that the CD of the gR-carotenoid complex is due to induced chirality of the carotenoid 10,11 . pH-dependent oligomer-monomer transition, and their CD spectra were reported by Demura et al 9 Recently it was reported that the pentameric form of the pigment predominant at pH 8 while the monomeric form at pH 3 15,33 . However, in presence of sal, the exact origin of the observed positive and negative CD lobes with the enhancement of intensity and reversal of the CD band sign compared to the native gR, is not clear.…”

“…The first possibility is based on excitonic interaction between sal and the retinal chromophore 21 . The second stems from the chiral conformation of the retinal in the presence of sal, while retinal-retinal excitonic coupling in a pigment pentamer form can be an additional cause for the CD spectrum 15,24,26,27 . Another possibility is based on a chiral conformation of sal in the presence of the retinal chromophore and nearby protein residues 19 .…”

A unique clade of light-driven proton-pumping rhodopsins evolved in the cyanobacterial lineage

“…The structure of N2098R was also compared with the cyanobacterial rhodopsin GR (PDB code 6NWD) 40 and ASR (PDB code 1XIO) 41 , which are included in the XLR and XeR rhodopsin clades, respectively. The overall structure of N2098R is similar to that of GR (RMSD 1.91 Å) and ASR (RMSD 1.40 Å) (Supporting Information Fig.…”

The Impact of Optogenetics on Regenerative Medicine

“…Unfortunately, several tissues located in the deepest zone cannot be investigated for clinical purposes. For this reason, researchers are studying alternative solutions, such as X-optogenetics, which involves X-rays, and U-optogenetics, which exploits sonoluminescence [51,52].…”