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Biophysical Journal Feb 2003Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more... (Comparative Study)
Comparative Study
Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more methionine residues undergo a structural change during the BR-->M portion of the BR photocycle. Selenomethionine was incorporated into BR using a cell-free protein translation system containing an amino acid mixture with selenomethionine substituted for methionine. BR-->M FTIR difference spectra recorded for unlabeled and selenomethionine-labeled cell-free expressed BR closely resemble the spectra of in vivo expressed BR. However, reproducible changes occur in two regions near 1,284 and 900 cm(-1) due to selenomethionine incorporation. Isotope labeled tyrosine was also co-incorporated with selenomethionine in order to confirm these assignments. Based on recent x-ray crystallographic studies, likely methionines which give rise to the FTIR difference bands are Met-118 and Met-145, which are located inside the retinal binding pocket and in a position to constrain the motion of retinal during photoisomerization. The assignment of methionine bands in the FTIR difference spectrum of BR provides a means to study methionine-chromophore interaction under physiological conditions. More generally, combining cell-free incorporations of selenomethionine into proteins with FTIR difference spectroscopy provides a useful method for investigating the role of methionines in protein structure and function.
Topics: Amino Acid Substitution; Bacteriorhodopsins; Cell-Free System; Methionine; Protein Conformation; Protein Folding; Selenomethionine; Spectroscopy, Fourier Transform Infrared; Staining and Labeling
PubMed: 12547777
DOI: 10.1016/S0006-3495(03)74912-1 -
Protein Science : a Publication of the... Jun 2018The conformational equilibria of integral membrane proteins have proven extremely difficult to characterize within native lipid bilayers. To circumvent technical issues,...
The conformational equilibria of integral membrane proteins have proven extremely difficult to characterize within native lipid bilayers. To circumvent technical issues, investigations of the structure and stability of α-helical membrane proteins are often carried out in mixed micelle or bicelle solvents that mimic the membrane and facilitate measurements of reversible folding. Under these conditions, the energetics of membrane protein folding are typically proportional to the mole fraction of an anionic detergent in the micelle. However, investigations of the folding and unfolding of bacteriorhodopsin (bR) surprisingly revealed that the folding rate is also highly sensitive to the bulk molar concentration of lipids and detergents. We show here that this rate enhancement coincides with changes in bicelle size and suggest this effect arises through restriction of the conformational search space during folding. In conjunction with previous mutagenic studies, these results provide additional evidence that a topological search limits the rate of bR folding. Furthermore, this finding provides insights into the manner by which micellar and bicellar environments influence the conformational stability of polytopic membrane proteins.
Topics: Bacteriorhodopsins; Lipid Bilayers; Micelles; Protein Conformation; Protein Folding; Thermodynamics
PubMed: 29604129
DOI: 10.1002/pro.3414 -
Structure (London, England : 1993) Sep 2009Bacteriorhodopsin and proteorhodopsin are simple heptahelical proton pumps containing a retinal chromophore covalently bound to helix G via a protonated Schiff base....
Bacteriorhodopsin and proteorhodopsin are simple heptahelical proton pumps containing a retinal chromophore covalently bound to helix G via a protonated Schiff base. Following the absorption of a photon, all-trans retinal is isomerized to a 13-cis conformation, initiating a sequence of conformational changes driving vectorial proton transport. In this study we apply time-resolved wide-angle X-ray scattering to visualize in real time the helical motions associated with proton pumping by bacteriorhodopsin and proteorhodopsin. Our results establish that three conformational states are required to describe their photocycles. Significant motions of the cytoplasmic half of helix F and the extracellular half of helix C are observed prior to the primary proton transfer event, which increase in amplitude following proton transfer. These results both simplify the structural description to emerge from intermediate trapping studies of bacteriorhodopsin and reveal shared dynamical principles for proton pumping.
Topics: Bacteriorhodopsins; Light; Protein Conformation; Rhodopsin; Rhodopsins, Microbial; Scattering, Radiation
PubMed: 19748347
DOI: 10.1016/j.str.2009.07.007 -
Protein Science : a Publication of the... Oct 2009Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli...
Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.
Topics: Bacteriorhodopsins; Cholates; Cholic Acids; Digitonin; Escherichia coli; Liposomes; Phosphatidylcholines; Protein Biosynthesis
PubMed: 19746358
DOI: 10.1002/pro.230 -
International Journal of Molecular... Dec 2022The use of photosensitive proteins has become a competitive solar energy solution, owing to its pollution-free nature, high conversion efficiency, and good...
The use of photosensitive proteins has become a competitive solar energy solution, owing to its pollution-free nature, high conversion efficiency, and good biocompatibility. Bacteriorhodopsin (bR) is an important light-sensitive protein that is widely used in the fabrication of photoelectronic devices. However, research on the optimization and comparison of the immobilization techniques is lacking. In this study, in order to obtain bR films with a high energy conversion efficiency, three immobilization techniques, namely dropcasting, electrophoretic sedimentation, and Langmuir-Blodgett deposition, were used to fabricate films, and their topographical and photoelectrical characteristics were compared. All three immobilization techniques can transfer bR molecules to substrates, forming functional photosensitive bR films. The absorption of the bR films at 568 nm reached the highest value of 0.3 under the EPS technique. The peak photocurrent for the EPS technique reached 5.03 nA. In addition, the EPS technique has the highest efficiency factor of 13.46, indicating that it can generate the highest value of photocurrent under the same light conditions, owing to the improved orientation, and no significant decrease in the peak photocurrent was observed after three weeks, which indicates the stability of the photoelectric response. These results indicate that the EPS technique has a great potential for the photoelectrical device fabrication and solar-energy conversion.
Topics: Bacteriorhodopsins
PubMed: 36555719
DOI: 10.3390/ijms232416079 -
Trends in Biotechnology Jul 1993Artificial retinas based on the light transducing photoelectric protein bacteriorhodopsin exhibit differential responsivity, edge enhancement and motion detection. Under...
Artificial retinas based on the light transducing photoelectric protein bacteriorhodopsin exhibit differential responsivity, edge enhancement and motion detection. Under appropriate conditions, these artificial receptors mimic the differential responsivity characteristic of mammalian photoreceptor cells. The use of orientated bacteriorhodopsin to generate the photoelectrical signal provides rapid responsivity, high quantum efficiency and offers the potential of directly coupling the protein response to charge-sensitive semiconductor arrays. The ability to manipulate the properties of the protein via chemical and genetic methods enhances design flexibility.
Topics: Animals; Artificial Organs; Bacteriorhodopsins; Biotechnology; Evaluation Studies as Topic; Humans; Models, Molecular; Photochemistry; Retina
PubMed: 7763952
DOI: 10.1016/0167-7799(93)90017-4 -
ACS Applied Materials & Interfaces Jun 2021Biomolecular devices based on photo-responsive proteins have been widely proposed for medical, electrical, and energy storage and production applications. Also,...
Biomolecular devices based on photo-responsive proteins have been widely proposed for medical, electrical, and energy storage and production applications. Also, bacteriorhodopsin (bR) has been extensively applied in such prospective devices as a robust photo addressable proton pump. As it is a membrane protein, in principle, it should function most efficiently when reconstituted into a fully fluid lipid bilayer, but in many model membranes, lateral fluidity of the membrane and protein is sacrificed for electrochemical addressability because of the need for an electroactive surface. Here, we reported a biomolecular photoactive device based on light-activated proton pump, bR, reconstituted into highly fluidic microcavity-supported lipid bilayers (MSLBs) on functionalized gold and polydimethylsiloxane cavity array substrates. The integrity of reconstituted bR at the MSLBs along with the lipid bilayer formation was evaluated by fluorescence lifetime correlation spectroscopy, yielding a protein lateral diffusion coefficient that was dependent on the bR concentration and consistent with the Saffman-Delbrück model. The photoelectrical properties of bR-MSLBs were evaluated from the photocurrent signal generated by bR under continuous and transient light illumination. The optimal conditions for a self-sustaining photoelectrical switch were determined in terms of protein concentration, pH, and light switch frequency of activation. Overall, a significant increase in the transient current was observed for lipid bilayers containing approximately 0.3 mol % bR with a measured photo-current of 250 nA/cm. These results demonstrate that the platforms provide an appropriate lipid environment to support the proton pump, enabling its efficient operation. The bR-reconstituted MSLB model serves both as a platform to study the protein in a highly addressable biomimetic environment and as a demonstration of reconstitution of seven-helix receptors into MSLBs, opening the prospect of reconstitution of related membrane proteins including G-protein-coupled receptors on these versatile biomimetic substrates.
Topics: Bacteriorhodopsins; Dimethylpolysiloxanes; Electrochemical Techniques; Gold; Light; Lipid Bilayers; Phosphatidylcholines; Phosphatidylethanolamines; Photochemical Processes; Unilamellar Liposomes
PubMed: 34121400
DOI: 10.1021/acsami.1c06798 -
Biochimica Et Biophysica Acta Aug 2000Recent advances in the determination of the X-ray crystallographic structures of bacteriorhodopsin, and some of its photointermediates, reveal the nature of the linkage... (Review)
Review
Recent advances in the determination of the X-ray crystallographic structures of bacteriorhodopsin, and some of its photointermediates, reveal the nature of the linkage between the relaxation of electrostatic and steric conflicts at the retinal and events elsewhere in the protein. The transport cycle can be now understood in terms of specific and well-described displacements of hydrogen-bonded water, and main-chain and side-chain atoms, that lower the pK(a)s of the proton release group in the extracellular region and Asp-96 in the cytoplasmic region. Thus, local electrostatic conflict of the photoisomerized retinal with Asp-85 and Asp-212 causes deprotonation of the Schiff base, and results in a cascade of events culminating in proton release to the extracellular surface. Local steric conflict of the 13-methyl group with Trp-182 causes, in turn, a cascade of movements in the cytoplasmic region, and results in reprotonation of the Schiff base. Although numerous questions concerning the mechanism of each of these proton (or perhaps hydroxyl ion) transfers remain, the structural results provide a detailed molecular explanation for how the directionality of the ion transfers is determined by the configurational relaxation of the retinal.
Topics: Bacteriorhodopsins; Crystallography; Energy Transfer; Ion Transport; Kinetics; Membrane Proteins; Models, Molecular; Molecular Conformation; Protein Conformation; Protein Transport; Retinaldehyde; X-Ray Diffraction
PubMed: 11004449
DOI: 10.1016/s0005-2728(00)00170-5 -
Nano Letters Feb 2015The role of the electron spin in chemistry and biology has received much attention recently owing to to the possible electromagnetic field effects on living organisms...
The role of the electron spin in chemistry and biology has received much attention recently owing to to the possible electromagnetic field effects on living organisms and the prospect of using molecules in the emerging field of spintronics. Recently the chiral-induced spin selectivity effect was observed by electron transmission through organic molecules. In the present study, we demonstrated the ability to control the spin filtering of electrons by light transmitted through purple membranes containing bacteriorhodopsin (bR) and its D96N mutant. The spin-dependent electrochemical cyclic voltammetry (CV) and chronoamperometric measurements were performed with the membranes deposited on nickel substrates. High spin-dependent electron transmission through the membranes was observed; however, after the samples were illuminated by 532 nm light, the spin filtering in the D96N mutant was dramatically reduced whereas the light did not have any effect on the wild-type bR. Beyond demonstrating spin-dependent electron transmission, this work also provides an interesting insight into the relationship between the structure of proteins and spin filtering by conducting electrons.
Topics: Bacteriorhodopsins; Electromagnetic Fields; Light
PubMed: 25621438
DOI: 10.1021/nl503961p -
Biophysical Journal Dec 1996The stationary electric dichroism of bacteriorhodopsin is in qualitative, but not quantitative, agreement with the orientation function for disks having a permanent...
The stationary electric dichroism of bacteriorhodopsin is in qualitative, but not quantitative, agreement with the orientation function for disks having a permanent dipole directed perpendicular to the plane and an induced dipole in the plane. Fits of the orientation function to data measured at low field strengths demonstrate: an increase of the permanent dipole moment mu with the square of the disk radius r2, whereas the polarizability alpha increases with r4; the ionic strength dependence is small for mu and clearly stronger for alpha; the permanent dipole moment is 4x10(6) D at r = 0.5 micron. According to the risetime constants, the induced dipole does not saturate and increases to 4x10(8) D at 40 kV/cm and r = 0.5 micron. The data indicate that the permanent dipole is not of some interfacial character but is due to a real assymetry of the charge distribution. The experimental dipole moment per protein monomer is approximately 55 D, whereas calculations based on the structure of Grigorieff et al. (Grigorieff, N., T.A. Ceska, K.H. Downing, J.M. Baldwin, and R. Henderson. 1996. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol. 259:393-421) provide a dipole moment of approximately 570 D. The difference is probably due to a nonsymmetric distribution of charged lipid residues. It is concluded that experimental dipole moments reflect the mu-potential at the plane of shear for rotational diffusion, in analogy to the sigma-potential used for translational diffusion. It is suggested that the permanent dipole of bacteriorhodopsin supports proton transport by attraction of protons inside and repulsion of protons outside of the cell. Dichroism rise curves at field strengths between E = 150 and 800 V/cm reveal an exponential component with time constants tau 3r in the range between 1 and 40 ms, which is not found in Brownian dynamics simulations on a disk structure using hydrodynamic and electric parameters characteristic of bacteriorhodopsin disks. The experimental data suggest that this process reflects a cooperative change of the bacteriorhodopsin structure, which is induced already at a remarkably low field strength of approximately 150 V/cm.
Topics: Bacteriorhodopsins; Electricity; Electrochemistry; Kinetics; Light; Mathematics; Models, Theoretical; Solutions; Static Electricity
PubMed: 8968607
DOI: 10.1016/S0006-3495(96)79531-0