Rhodopsins

The sequenced genome of the poly-extremophile Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa (3,570 m), a High-Altitude Andean Lake (HAAL) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area is exposed to the highest UV irradiation on Earth, making the microbial community living in the stromatolites test cases for survival strategies under extreme conditions. The heterologous expressed protein E17R from Exiguobacterium (248 amino acids, 85% sequence identity to its ortholog ESR from E. sibiricum) was assembled with retinal displaying an absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-subfamily. Titration down to low pH values (eventually causing partial protein denaturation) indicated a pK value between two and three. Global fitting of data from laser flash-induced absorption changes gave evidence for an early red-shifted intermediate (its formation being below the experimental resolution...

Detergent-resistant membrane microdomains in the plasma membrane, known as lipid rafts, have been implicated in various cellular processes. We report here that a low-density Triton X-100-insoluble membrane (detergent-resistant membrane; DRM) fraction is present in bovine rod photoreceptor outer segments (ROS). In dark-adapted ROS, transducin and most of cGMPphosphodiesterase (PDE) were detergent-soluble. When ROS membranes were exposed to light, however, a large portion of transducin localized in the DRM fraction. Furthermore, on addition of guanosine 5-3-O-(thio)triphosphate (GTP␥S) to light-bleached ROS, transducin became detergent-soluble again. PDE was not recruited to the DRM fraction after light stimulus alone, but simultaneous stimulation by light and GTP␥S induced a massive translocation of all PDE subunits to the DRM. A cholesterol-removing reagent, methyl-␤-cyclodextrin, selectively but partially solubilized PDE from the DRM, suggesting that cholesterol contributes, at least in part, to the association of PDE with the DRM. By contrast, transducin was not extracted by the depletion of cholesterol. These data suggest that transducin and PDE are likely to perform their functions in phototransduction by changing their localization between two distinct lipid phases, rafts and surrounding fluid membrane, on disc membranes in an activation-dependent manner.

Photochemical reaction dynamics of the primary events in recombinant bacteriorhodopsin (bRrec) was studied by
femtosecond laser absorption spectroscopy with 25-fs time resolution. bRrec was produced in an Escherichia coli expression system. Since bRrec was prepared in a DMPC–CHAPS micelle system in the monomeric form, its comparison with trimeric and monomeric forms of the native bacteriorhodopsin (bRtrim and bRmon, respectively) was carried out. We found that bRrec intermediate I (excited state of bR) was formed in the range of 100 fs, as in the case of bRtrim and bRmon. Further processes, namely the decay of the excited state I and the formation of intermediates J and K of bRrec, occurred more slowly compared to bRtrim, but similarly to bRmon. The lifetime of intermediate I, judging from the signal of ΔAESA(470480 nm), was 0.68 ps (78%) and 4.4 ps (22%) for bRrec, 0.52 ps (73%) and 1.7 ps (27%) for bRmon, and 0.45 ps (90%) and 1.75 ps (10%) for bRtrim. The formation time of intermediate K, judging from the signal of ΔAGSA(625635 nm), was 13.5 ps for bRrec, 9.8 ps for bRmon, and 4.3 ps for bRtrim. In addition, there was a decrease in the photoreaction efficiency of bRrec and bRmon as seen by a decrease in absorbance in the differential spectrum of the intermediate K by ∼14%. Since photochemical properties of bRrec are similar to those of the monomeric form of the native protein, bRrec and its mutants can be considered as a basis for further studies of the mechanism of bacteriorhodopsin functioning.

Since algal rhodopsins, the eukaryotic seven-transmembrane proteins, are generally difficult to express in Escherichia coli, eukaryotic cells have been used for heterologous expression. Mistic, a membrane-associated protein that was originally discovered in Bacillus subtilis, has been shown to improve the expression levels of many foreign integral membrane proteins in E. coli when used as a fusion partner linked to the N-terminus of cargo proteins.
Here, we expressed two algal rhodopsins with N- and C-terminal Mistic domains in E. coli - Acetabularia rhodopsin I (ARI) and Chlamydomonas sensory rhodopsin B (CSRB, channel rhodopsin 2). UV/VIS spectroscopy, pH titration of proton acceptor residue, laser-induced photolysis and electrophysiological measurement were used for investigating important residues in proton transport and spectroscopic characters of the proteins.
Protein yield of two algal rhodopsins was enhanced, obtaining 0.12 mg of Mistic-ARI and 0.04 mg of Mistic-CSRB per liter of culture. Spheroplasts expression Mistic-ARI had outward proton-pumping activity, indicating protein functionality. Asp89 of ARI changed its protonation state by light absorption, and Asp100 was important for O(600) formation. Electrophysiology revealed that both residues took part in proton transport. The spectroscopic analyses of Mistic-CSRB revealed its characteristics.
Fusion to the membrane-integrating protein Mistic can enhance overexpression of eukaryotic type I rhodopsins in E. coli.
These findings indicate that Mistic fusion and E. coli expression method could be an effective, low cost technique for studying eukaryotic membrane proteins. This may have useful implications, for example, in studying structural characteristics and optogenetics for rhodopsins.

We cloned an intronless, nuclear-encoded opsin gene from an EST library of Acetabularia acetabulum. Acetabularia rhodopsin I (ARI) encodes a protein of 246 amino acids with molecular weight of 27 kDa. ARI was reconstituted in the Xenopus oocyte expression system to characterize its electrophysiological properties utilizing the two-electrode voltage-clamping technique. Oocytes where ARI cRNA was injected displayed outward directed currents in response to light. The maximum action spectrum of ARI was detected at 520 nm green light. Light-stimulated ARI current amplitude was altered by the protons, but not by the other ions in recording solutions, suggesting that the algal rhodopsin is a light-stimulated proton pump. Typical proton-mediated outward current elicited by 520 nm light was characterized with two phases of non-inactivating outward current following initial transient current. Taken together, we here reported cloning of a novel Acetabularia opsin gene which was characterized to be a proton-pump stimulated by light.

The sequenced genome of the poly-extremophile Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa (3,570 m), a High-Altitude Andean Lake (HAAL) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area is exposed to the highest UV irradiation on Earth, making the microbial community living in the stromatolites test cases for survival strategies under extreme conditions. The heterologous expressed protein E17R from Exiguobacterium (248 amino acids, 85% sequence identity to its ortholog ESR from E. sibiricum) was assembled with retinal displaying an absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-subfamily. Titration down to low pH values (eventually causing partial protein denaturation) indicated a pK value between two and three. Global fitting of data from laser flash-induced absorption changes gave evidence for an early red-shifted intermediate (its formation being below the experimental resolution...

The sequenced genome of the poly-extremophile Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa (3,570 m), a High-Altitude Andean Lake (HAAL) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area is exposed to the highest UV irradiation on Earth, making the microbial community living in the stromatolites test cases for survival strategies under extreme conditions. The heterolo-gous expressed protein E17R from Exiguobacterium (248 amino acids, 85% sequence identity to its ortholog ESR from E. sibiricum) was assembled with retinal displaying an absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-sub-family. Titration down to low pH values (eventually causing partial protein denaturation) indicated a pK value between two and three. Global fitting of data from laser flash-induced absorption changes gave evidence for an early red-shifted intermediate (its formation being below the experimental resolution) that decayed (τ 1 = 3.5 μs) into another red-shifted intermediate. This species decayed in a two-step process (τ 2 = 84 μs, τ 3 = 11 ms), to which the initial state of E17-PR was reformed with a kinetics of 2 ms. Proton transport capability of the HAAL protein was determined by BLM measurements. Additional blue light irradiation reduced the proton current, clearly identifying a blue light absorbing, M-like intermediate. The apparent absence of this intermediate is explained by closely matching formation and decay kinetics.