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Highly expressible bacteriorhodopsin (HEBR) is a light-triggered protein (optogenetic protein) that has seven transmembrane regions with retinal bound as their chromophore to sense light. HEBR has controllable photochemical properties and regulates activity on proton pumping. In this study, we generated HEBR protein and incubated with lung cancer cell lines (A549 and H1299) to evaluate if there was a growth-inhibitory effect with or without light illumination. The data revealed that the HEBR protein suppressed cell proliferation and induced the G 0 /G 1 cell cycle arrest without light illumination. Moreover, the migration abilities of A549 and H1299 cells were reduced by ~17% and ~31% after incubation with HEBR (40 μg/mL) for 4 h. The Snail-1 gene expression level of the A549 cells was significantly downregulated by ~50% after the treatment of HEBR. In addition, HEBR significantly inhibited the gene expression of Sox-2 and Oct-4 in H1299 cells. These results suggested that the HEBR protein may inhibit cell proliferation and cell cycle progression of lung cancer cells, reduce their migration activity, and suppress some stemness-related genes. These findings also suggested the potential of HEBR protein to regulate the growth and migration of tumor cells, which may offer the possibility for an anticancer drug.

Fullerenes have unique biocompatibility and photoelectric properties and are candidate materials for biomedical applications. Several cell membrane proteins in nature such as bacteriorhodopsin also have photoelectric properties. Highly expressible bacteriorhodopsin (HEBR) is a novel light-sensitive opsin that has the potential to trigger neural activities through optogenetic modulation. Here, HEBR plasmids are delivered to human fibroblasts and the cells are exposed to C60 fullerene self-assembled 2D nanosheets. Results show that the above approach combined with light stimulation (3 s duration and three times per day) may promote reprogramming and differentiation of human fibroblasts into neural-like cells in 7 d without any neural induction medium. The special photoelectric properties of fullerenes as culture substrates and transfected HEBR on the cell membrane may provide a new optogenetic platform for regulating the location (C60 nanosheet) and time (frequency of light illumination) for human fibroblasts to become neural-like cells, and may be applied to improve neural regeneration in the future., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cubic phase of monoolein (MO) has successfully been used for crystallization of membrane proteins. It is likely that the transition to a lamellar phase upon dehydration is important for the crystallization process, and that the internal dimensions of the lipid phases (i.e., water pore diameter) are crucial for the inclusion and the diffusion of membrane proteins. In the present study, we investigated the cubic-to-lamellar phase transitions in the MO-water and the MO-distearoyl phosphatidyl glycerol (DSPG) systems. The MO-water system was investigated by means of isothermal sorption and desorption microcalorimetry. We show that the transition from cubic to lamellar phase induced by desorption is driven by entropy. At 25 degrees C, this occurs at a water activity of 0.98 with a transition enthalpy of 860 J/mol (MO). The phase behavior was also investigated in the presence of a small amount of the transmembrane protein bacteriorhodopsin (bR), and a detergent, octyl glucoside (OG), and it was shown that both bR and OG stabilize the lamellar phase. Analogous results were obtained for the MO-DSPG-water system. The latter system resembles the MO-water system in that a cubic-to-lamellar phase transition is induced by dehydration, although the structural properties of these phases are slightly different. Finally, we demonstrate that bR can be crystallized from a cubic phase of MO-DSPG-buffer.

The sophistication of supported lipid bilayer membranes has increased steadily as new applications are being explored. In general, tethered lipids are used to anchor the lipid bilayer to the substrate. Here we describe a new type of anchoring system for supported lipid bilayers that is based on biotin-PEG3400-bacteriorhodopsin conjugates. Amine-based coupling was used to construct the polymer conjugates, followed by fluorophore labeling to enable confocal imaging. The bacteriorhodopsin-based anchoring system was used to construct solid-supported vesicles from streptavidin-coated microspheres. This method could provide a new route for the stability enhancement of supported lipid bilayer membrane assemblies.

Active accumulation of neurotransmitters by synaptic vesicles is an essential component of the synaptic transmission cycle. Isolated vesicles show energy-dependent uptake of several transmitters by processes which are apparently mediated by a proton electrochemical potential across the vesicle membrane. Although this energy gradient is probably generated by a proton ATPase, the functional separation of ATP cleavage and transmitter uptake activity has only been shown clearly for monoamine transport. We report here that the light-driven proton pump, bacteriorhodopsin, can replace the endogenous proton ATPase in proteoliposomes reconstituted from vesicular detergent extracts. The system shows light-dependent uptake of glutamate with properties very similar to those observed in intact vesicles, e.g. chloride dependence or stimulation by NH4+. Our experiments show that the proton pump and the glutamate transporter are separate entities and provide a powerful tool for further characterization of the glutamate carrier.

The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 mumol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.

Several spectroscopic techniques (absorption, emission, transient absorption and differential scanning calorimetry--DSC) were used to investigate the deprotonation of dibucaine.HCl in a hydrophobic environment, and the interaction sites and mechanisms of the local anesthetic dibucaine.HCl on bacteriorhodopsin (bR) in purple membrane. The important results are summarized as follows: (1) the visible absorption features of native (lambda max = 568 nm) and deionized (lambda max = 608 nm) bR are sensitive to the amount of dibucaine.HCl added; (2) the emission spectrum of dibucaine.HCl embedded in the retinal-free mutant bR is similar to that of dibucaine free base in Triton X-100 micellar solutions; (3) the phosphorescence emission of dibucaine at 77 K is completely quenched by bR and the fluorescence quenching rate for the incorporated dibucaine.HCl in bR was determined as kq = 4.09 x 10(13) M-1 s-1; (4) the incorporation of dibucaine.HCl in bR inhibits the slow component rate of formation of M412 and decreases the amount of M412 formation in the photochemical cycle of bR; and (5) the thermal stability of native bR was measured by DSC in the presence and absence of dibucaine and yielded an endothermic transition at 95.9 +/- 1.0 degrees C with 13.6 J/g (3.25 +/- 0.12 cal/g) of enthalpy changes. All observations suggest that the action site of the local anesthetic, dibucaine.HCl, is near or at the chromophore, i.e. the retinal Schiff base of bR. The anesthetic action on bR purple membrane is probably via a specific site binding, but not a conformational mechanism.