Liquid crystal (LC) biosensors have received significant attention for their potential applications for point-of-care devices due to their sensitivity, low cost, and easy read-out. They have been employed to detect a wide range of important biological molecules. However, detecting the function of membrane proteins has been extremely challenging due to the difficulty of integrating membrane proteins, lipid membranes, and LCs into one system. In this study, we addressed this challenge by monitoring the proton-pumping function of bacteriorhodopsin (bR) using a pH-sensitive LC thin film biosensor. To achieve this, we deposited purple membranes (PMs) containing a 2D crystal form of bRs onto an LC-aqueous interface. Under light, the PM patches changed the local pH at the LC-aqueous interface, causing a color change in the LC thin film that is observable through a polarizing microscope with crossed polarizers. These findings open up new opportunities to study the biofunctions of membrane proteins and their induced local environmental changes in a solution using LC biosensors.

The article correlates between symmetry breaking and phase transition. An analogy, extending from physics to biology, is known to exist between these two topics. Bacteriorhodopsin (bR) as a paradigm of membrane proteins has been used as a case study in the present work. The bR, as the sole protein embedded in what is called a purple membrane (PM), has attracted widespread interest in bionanotechnological applications. The lipids of PM have a crucial role in maintaining the crystal lattice of bR inside PM. For this reason, the present work has been concerned with elucidating the thermal phase transition properties of the PM lipids in orthogonal directions. The results indicated that the axial symmetry of bR exhibits considerable changes occurring at the thermal phase transition of lipids. These changes are brought by an anomaly observed in the time course of orthogonal electric responses during the application of thermal fields on PM. The observed anomaly may bear on symmetry breaking in bR occurring at the phase transition of lipids based on such analogy found between symmetry breaking and phase transition. Lipid-protein interactions may underlie the broken axial symmetry of bR at such lipid thermal transition of PM. Accordingly, thermally perturbed axial symmetry of bR may be of biological relevance relying on the essence of the crystal lattice of bR. Most importantly, a question has to be raised in the present study: Can bR, as a helical protein with broken axial symmetry, affect the symmetry breaking of helical light? This may be of potential technical applications based on a recent discovery that bR breaks the symmetry of helical light.

It marked half a century since the discovery of bacteriorhodopsin two years ago. On this occasion, I have revisited historically important diffraction studies of this membrane protein, based on my recollections. X-ray diffraction and electron diffraction, and electron microscopy, described the low-resolution structure of bacteriorhodopsin within the purple membrane. Neutron diffraction was effective to assign the helical regions in the primary structure with 7 rods revealed by low-resolution structure as well as to describe the retinal position. Substantial conformational changes upon light illumination were clarified by the structures of various photointermediates. Early trials of time-resolved studies were also introduced. Models for the mechanism of light-driven proton pump based on the low-resolution structural studies are also described. Significantly, they are not far from the today\'s understanding. I believe that the spirit of the early research scientists in this field and the essence of their studies, which constitute the foundations of the field, still actively fertilizes current membrane protein research.

Native mass spectrometry (MS) was used to detect the membrane protein, bacteriorhodopsin (bR), in its 27 kDa monomeric form and trimeric assemblies directly from lipid-containing purple membranes (PMs) from the halophilic archaeon, Halobacterium salinarum. Trimer bR ion populations bound to lipid molecules were detected with n-octyl β-d-glucopyranoside as the solubilizing detergent; the use of octyl tetraethylene glycol monooctyl ether or n-dodecyl-β-d-maltopyranoside resulted in only detection of monomeric bR. The archaeal lipids phosphotidylglycerolphosphate methyl ester and 3-HSO3-Galp-β1,6-Manp-α1,2-Glcp-α1,1-sn-2,3-diphytanylglycerol were the only lipids in the PMs found to bind to bR, consistent with previous high-resolution structural studies. Removal of the lipids from the sample resulted in the detection of only the bR monomer, highlighting the importance of specific lipids for stabilizing the bR trimer. To the best of our knowledge, this is the first report of the detection of the bR trimer with resolved lipid-bound species by MS.

An effective early diagnosis is important for rheumatoid arthritis (RA) management. This study reveals a novel RA detection method using bacteriorhodopsin as a photoelectric transducer, a light-driven proton pump in purple membranes (PMs). It was devised by covalently conjugating a PM monolayer-coated electrode with a citrullinated-inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3)542-556 peptide that recognizes the serum RA-associated autoantibodies. The direct serum coating decreased the photocurrents in the biosensor, with the reduction in the photocurrent caused by coating with an RA-patient serum that is significantly larger than that with a healthy-control serum (38.1% vs. 20.2%). The difference in the reduction in the photocurrent between those two serum groups widened after the serum-coated biosensor was further labeled with gold nanoparticle (AuNP)-conjugated anti-IgA (anti-IgA-AuNP) (53.6% vs. 30.6%). Both atomic force microscopic (AFM) and Raman analyses confirmed the sequential peptide, serum, and anti-IgA-AuNP coatings on the PM-coated substrates. The reductions in the photocurrent measured in both the serum and anti-IgA-AuNPs coating steps correlated well with the results using commercial enzyme-linked immunosorbent assay kits (Spearman rho = 0.805 and 0.787, respectively), with both a sensitivity and specificity close to 100% in both steps. It was shown that an RA diagnosis can be performed in either a single- or two-step mode using the developed biosensor.

A uniformly oriented purple membrane (PM) monolayer containing photoactive bacteriorhodopsin has recently been applied as a sensitive photoelectric transducer to assay color proteins and microbes quantitatively. This study extends its application to detecting small molecules, using adenosine triphosphate (ATP) as an example. A reverse detection method is used, which employs AuNPs labeling and specific DNA strand displacement. A PM monolayer-coated electrode is first covalently conjugated with an ATP-specific nucleic acid aptamer and then hybridized with another gold nanoparticle-labeled nucleic acid strand with a sequence that is partially complementary to the ATP aptamer, in order to significantly minimize the photocurrent that is generated by the PM. The resulting ATP-sensing chip restores its photocurrent production in the presence of ATP, and the photocurrent recovers more effectively as the ATP concentration increases. Direct and single-step ATP detection is achieved in 15 min, with detection limits of 5 nM and a dynamic range of 5 nM-0.1 mM. The sensing chip exhibits high selectivity against other ATP analogs and is satisfactorily stable in storage. The ATP-sensing chip is used to assay bacterial populations and achieves a detection limit for Bacillus subtilis and Escherichia coli of 102 and 103 CFU/mL, respectively. The demonstration shows that a variety of small molecules can be simultaneously quantified using PM-based biosensors.

Recently in this Journal, James Lee employed his transmembrane electrostatically localized proton (TELP) hypothesis and the notion of a transient protonic capacitor to explain the force holding protons at the surface of bacteriorhodopsin purple membrane fragments. Here we show that purple membrane fragments cannot maintain the requisite transient non-zero transmembrane potential, and even if they could, it would not support the surface proton current moving from the P side to the N side that was reported by Heberle et al. (Nature, 1994). Currently accepted models explain the force keeping protons at the membrane surface by invoking the unusual structure of water at the interface which serves to stabilize the proton (energy well) and/or raise the activation ∆G‡ (energy barrier) for release to the bulk phase. Any future invocations of TELP should be required to include experimental measurements carried out at the surfaces of lipid bilayer membranes and/or biological membranes.

Bacteriorhodopsin (bR) of purple membrane (PM) is a retinal protein that forms aggregates in the form of trimers constituting, together with archaeal lipids, the crystalline structure of PM. The rotary motion of bR inside PM may be pertinent in understanding the essence of the crystalline lattice. An attempt has been made to determine the rotation of bR trimers which has been found to be detected solely at thermal phase transitions of PM, namely lipid, crystalline lattice and protein melting phase transitions. The temperature dependences of dielectric versus electronic absorption spectra of bR have been determined. The results suggest that the rotation of bR trimers, together with concomitant bending of PM, are most likely brought by structural changes in bR which might be driven by retinal isomerization and mediated by lipid. The rupturing of the lipid-protein contact might consequently lead to rotation of trimers associated with bending, curling or vesicle formation of PM. So the retinal reorientation may underlie the concomitant rotation of trimers. Most importantly, rotation of trimers might play a role, in terms of the essence of the crystalline lattice, in the functional activity of bR and may serve physiological relevance.

In the present study, the dependency of purple membrane (PM) dielectric responses on the wavelength of light in the range 380-750 nm has showed meaningful changes about the rotation of PM in suspension and about the rotation of bacteriorhodopsin (bR) trimer inside PM, as well. The action spectrum of PM random walk substantiates the existence of two states of bR. One of them (blue edge-state) lies at the blue edge and the other (red edge-state) at the red edge of the visible absorption of bR. The results might bear on correlation of these bands to some bR photocycle intermediates or bR photoproducts. The results implicate the protein-chromophore interactions that eventually underlie protein-lipid interactions. Disrupting the protein-lipid contact during the illumination with light of wavelength in ranges of (410-470 nm) and (610-720 nm) has resulted in emergence of distinct dielectric dispersion at 0.06-0.08 MHz which is comparable to the size of bR trimer or monomer.The work reports on the chromatic adaptation of bR in view of the dielectric spectral parameters of PM. It aimed to explore a correlation seemingly found between the light wavelength and the relaxations of bR trimer inside PM. Changes in rotational diffusion of bR trimer upon blue and red light illumination can influence the three dimensional data storage based on bR, which may implicate bR in bioelectronics.

In order to elucidate the old, still unsolved problem of how the diffuse electric double layer responds to an abrupt, intramolecular charge displacement inside a biological membrane, we investigated the fastest components of the light-induced electric signals of bacteriorhodopsin and its mutants, in numerous ionic and buffer solutions. The obtained data for temperature and solute concentration dependence were interpreted as a consequence of changes in the capacity of the diffuse double layer surrounding the purple membrane. The possible physiological consequences of this so far not demonstrated phenomenon are discussed.