Bacteriorhodopsin and the purple membrane of halobacteria

Biological ion pumps, such as bacteriorhodopsin (bR), utilize photons to move ions against concentration gradients, offering energy harvesting and spatiotemporal control of chemical gradients. This capability goes far beyond the capabilities of today's synthetic devices, suggesting a hybrid approach to embed bRs in synthetic devices in order to direct the proton flow towards useful system applications. In this study, a hybrid silicon-based nanochannel network with integrated purple membranes (PM) containing bR was fabricated. The fabrication method combines thermal scanning probe lithography, etching techniques, atomic layer deposition, plasma-enhanced chemical vapor deposition, and photolithography to create devices with buried nanochannels on silicon substrates. PM patches were deposited onto specified sites by a tunable nanofluidic confinement apparatus. The resulting device holds the potential for locally controlling directed ion transport in micrometer scale devices, a first step towards applications, such as locally affected proton catalyzed chemical reaction networks. Furthermore, this fabrication strategy, employing a maskless overlay, is a tool for constructing intricate nanofluidic network designs which are mechanically robust and straightforward to fabricate.

The most effective tested optogenetic tools available for neuronal silencing are the light-gated anion channel proteins found in the cryptophyte alga Guillardia theta (GtACRs). Molecular mechanisms of GtACRs, including the photointermediates responsible for the open channel state, are of great interest for understanding their exceptional conductance. In this study, the photoreactions of GtACR1 and its D234N, A75E, and S97E mutants were investigated using multichannel time-resolved absorption spectroscopy. For each of the proteins, the analysis showed two early microsecond transitions between K-like and L-like forms and two late millisecond recovery steps. Spectral forms associated with potential molecular intermediates of the proteins were derived and their evolutions in time were analyzed. The results indicate the presence of isospectral intermediates in the photocycles and expand the range of potential intermediates responsible for the open channel state.

Photosensitive proteins are extremely interesting, since they can regulate many functional processes in living cells, through the change of their structure in response to light absorption. This activation produces processes such as photosynthesis and phototaxis, or biomass, which can be used as renewable form of energy. Moreover, optogenetics and superresolution fluorescence microscopy approaches have triggered the generation of genetically modified photosensitive proteins. Due to these implications, the characterization of photosensitive proteins and their functioning is of crucial importance in real life. This review aims to collect the fundamental theoretical issues and the prominent advances in dealing with photosensitive proteins, with particular attention to the modeling of the behavior and organization of these molecules in the realm of Proteotronics. Together with the most interesting results, we recall some computational tools developed to achieve these results. The overview is completed by some open questions that emerge in this field of research.

The proton pumping cycle of bacteriorhodopsin (bR) is initiated when the retinal chromophore with the 13-trans configuration is photo-isomerized into the 13-cis configuration. To understand the recovery processes of the initial retinal configuration that occur in the late stage of the photocycle, we have performed a comprehensive analysis of absorption kinetics data collected at various pH levels and at different salt concentrations. The result of analysis revealed the following features of the late stages of the trans photocycle. i) Two substates occur in the O intermediate. ii) The visible absorption band of the first substate (O1) appears at a much shorter wavelength than that of the late substate (O2). iii) O1 is in rapid equilibrium with the preceding state (N), but O1 becomes less stable than N when an ionizable residue (X₁) with a pKa value of 6.5 (in 2 M KCl) is deprotonated. iv) At a low pH and at a low salt concentration, the decay time constant of O2 is longer than those of the preceding states, but the relationship between these time constants is altered when the medium pH or the salt concentration is increased. On the basis of the present observations and previous studies on the structure of the chromophore in O, we suspect that the retinal chromophore in O1 takes on a distorted 13-cis configuration and the O1-to-O2 transition is accompanied by cis-to-trans isomerization about C13C14 bond.

The surface charge of brain endothelial cells forming the blood-brain barrier (BBB) is highly negative due to phospholipids in the plasma membrane and the glycocalyx. This negative charge is an important element of the defense systems of the BBB. Lidocaine, a cationic and lipophilic molecule which has anaesthetic and antiarrhytmic properties, exerts its actions by interacting with lipid membranes. Lidocaine when administered intravenously acts on vascular endothelial cells, but its direct effect on brain endothelial cells has not yet been studied. Our aim was to measure the effect of lidocaine on the charge of biological membranes and the barrier function of brain endothelial cells. We used the simplified membrane model, the bacteriorhodopsin (bR) containing purple membrane of Halobacterium salinarum and culture models of the BBB. We found that lidocaine turns the negative surface charge of purple membrane more positive and restores the function of the proton pump bR. Lidocaine also changed the zeta potential of brain endothelial cells in the same way. Short-term lidocaine treatment at a 10 μM therapeutically relevant concentration did not cause major BBB barrier dysfunction, substantial change in cell morphology or P-glycoprotein efflux pump inhibition. Lidocaine treatment decreased the flux of a cationic lipophilic molecule across the cell layer, but had no effect on the penetration of hydrophilic neutral or negatively charged markers. Our observations help to understand the biophysical background of the effect of lidocaine on biological membranes and draws the attention to the interaction of cationic drug molecules at the level of the BBB.

The photosensitive protein bacteriorhodopsin (bR) has been shown to be a promising material for optoelectronic applications, but it cannot effectively absorb and utilize light energy in the near-infrared (NIR) region of the optical spectrum. Semiconductor quantum dots (QDs) have two-photon absorption cross-sections two orders of magnitude larger than those of bR and can effectively transfer the up-converted energy of two NIR photons to bR via the Förster resonance energy transfer (FRET). In this study, we have engineered a photoelectrochemical cell based on a hybrid material consisting of QDs and bR-containing purple membranes (PMs) of Halobacterium salinarum and demonstrated that this cell can generate an electrical signal under the two-photon laser excitation. We have shown that the efficiency of light conversion by the PM–QD hybrid material under two-photon excitation is up to 4.3 times higher than the efficiency of conversion by PMs alone. The QD integration into the bR-containing PMs significantly improves the bR capacity for utilizing light upon two-photon laser excitation, thus paving the way to the engineering of biologically inspired hybrid NIR nonlinear optoelectronic elements. The nonlinear nature of two-photon excitation may provide considerable advantages, such as a sharp sensitivity threshold and the possibility of precise three-dimensional location of excitation in holography and optical computing.