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Journal of the American Chemical Society Jan 2019Bacteriorhodopsin represents the simplest, and possibly most abundant, phototropic system requiring only a retinal-bound transmembrane protein to convert photons of...
Bacteriorhodopsin represents the simplest, and possibly most abundant, phototropic system requiring only a retinal-bound transmembrane protein to convert photons of light to an energy-generating proton gradient. The creation and interrogation of a microbial rhodopsin mimic, based on an orthogonal protein system, would illuminate the design elements required to generate new photoactive proteins with novel function. We describe a microbial rhodopsin mimic, created using a small soluble protein as a template, that specifically photoisomerizes all- trans to 13- cis retinal followed by thermal relaxation to the all- trans isomer, mimicking the bacteriorhodopsin photocycle, in a single crystal. The key element for selective isomerization is a tuned steric interaction between the chromophore and protein, similar to that seen in the microbial rhodopsins. It is further demonstrated that a single mutation converts the system to a protein photoswitch without chromophore photoisomerization or conformational change.
Topics: Bacteriorhodopsins; Biomimetics; Light; Models, Molecular; Movement; Protein Conformation; Stereoisomerism; Temperature
PubMed: 30580520
DOI: 10.1021/jacs.8b12493 -
Bioelectrochemistry (Amsterdam,... Aug 2022In order to elucidate the old, still unsolved problem of how the diffuse electric double layer responds to an abrupt, intramolecular charge displacement inside a...
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.
Topics: Bacteriorhodopsins; Cell Membrane; Electricity; Light; Temperature
PubMed: 35487144
DOI: 10.1016/j.bioelechem.2022.108138 -
Biochimica Et Biophysica Acta.... Oct 2022The proton pumping cycle of bacteriorhodopsin (bR) is initiated when the retinal chromophore with the 13-trans configuration is photo-isomerized into the 13-cis...
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 (X) 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.
Topics: Bacteriorhodopsins; Hydrogen-Ion Concentration; Kinetics
PubMed: 35753392
DOI: 10.1016/j.bbamem.2022.183998 -
Biophysical Journal Apr 2008We studied the low-frequency terahertz spectroscopy of two photoactive protein systems, rhodopsin and bacteriorhodopsin, as a means to characterize collective... (Comparative Study)
Comparative Study
We studied the low-frequency terahertz spectroscopy of two photoactive protein systems, rhodopsin and bacteriorhodopsin, as a means to characterize collective low-frequency motions in helical transmembrane proteins. From this work, we found that the nature of the vibrational motions activated by terahertz radiation is surprisingly similar between these two structurally similar proteins. Specifically, at the lowest frequencies probed, the cytoplasmic loop regions of the proteins are highly active; and at the higher terahertz frequencies studied, the extracellular loop regions of the protein systems become vibrationally activated. In the case of bacteriorhodopsin, the calculated terahertz spectra are compared with the experimental terahertz signature. This work illustrates the importance of terahertz spectroscopy to identify vibrational degrees of freedom which correlate to known conformational changes in these proteins.
Topics: Bacteriorhodopsins; Computer Simulation; Light; Microwaves; Models, Chemical; Models, Molecular; Protein Conformation; Rhodopsin; Spectrophotometry, Infrared
PubMed: 18199669
DOI: 10.1529/biophysj.107.105163 -
Biochimica Et Biophysica Acta Jul 2004In a light-driven proton-pump protein, bacteriorhodopsin (BR), protonated Schiff base of the retinal chromophore and Asp85 form ion-pair state, which is stabilized by a... (Review)
Review
In a light-driven proton-pump protein, bacteriorhodopsin (BR), protonated Schiff base of the retinal chromophore and Asp85 form ion-pair state, which is stabilized by a bridged water molecule. After light absorption, all-trans to 13-cis photoisomerization takes place, followed by the primary proton transfer from the Schiff base to Asp85 that triggers sequential proton transfer reactions for the pump. Fourier transform infrared (FTIR) spectroscopy first observed O-H stretching vibrations of water during the photocycle of BR, and accurate spectral acquisition has extended the water stretching frequencies into the entire stretching frequency region in D(2)O. This enabled to capture the water molecules hydrating with negative charges, and we have identified the water O-D stretch at 2171 cm(-1) as the bridged water interacting with Asp85. We found that retinal isomerization weakens the hydrogen bond in the K intermediate, but not in the later intermediates such as L, M, and N. On the basis of the observation particularly on the M intermediate, we proposed a model for the mechanism of proton transfer from the Schiff base to Asp85. In the "hydration switch model", hydration of a water molecule is switched in the M intermediate from Asp85 to Asp212. This will have raised the pK(a) of the proton acceptor, and the proton transfer is from the Schiff base to Asp85.
Topics: Bacteriorhodopsins; Crystallography, X-Ray; Halobacterium salinarum; Hydrogen Bonding; Models, Molecular; Proton Pumps; Schiff Bases; Spectroscopy, Fourier Transform Infrared; Water
PubMed: 15282177
DOI: 10.1016/j.bbabio.2004.03.015 -
Biochemistry. Biokhimiia Nov 2001The function of bacteriorhodopsin in Halobacterium salinarum is to pump protons from the internal side of the plasma membrane to the external after light excitation,... (Review)
Review
The function of bacteriorhodopsin in Halobacterium salinarum is to pump protons from the internal side of the plasma membrane to the external after light excitation, thereby building up electrochemical energy. This energy is transduced into biological energy forms. This review deals with one of the methods elaborated for recording the charge transfer inside the protein. In this method the current produced in oriented purple membrane containing bacteriorhodopsin is measured. It is shown that this method might be applied not only to correlate charge motion with the photocycle reactions but also for general problems like effect of water, electric field, and different ions and buffers for the functioning of proteins.
Topics: Bacteriorhodopsins; Halobacterium; Photochemistry; Protons
PubMed: 11743868
DOI: 10.1023/a:1013179101782 -
Proceedings of the National Academy of... Dec 2017Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but...
Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but has remained mostly silent to protonation changes in the aqueous medium. Here, by selectively monitoring vibrational changes of buffer molecules with a temporal resolution of 6 µs, we have traced proton release and uptake events in the light-driven proton-pump bacteriorhodopsin and correlate these to other molecular processes within the protein. We demonstrate that two distinct chemical entities contribute to the temporal evolution and spectral shape of the continuum band, an unusually broad band extending from 2,300 to well below 1,700 cm The first contribution corresponds to deprotonation of the proton release complex (PRC), a complex in the extracellular domain of bacteriorhodopsin where an excess proton is shared by a cluster of internal water molecules and/or ionic E194/E204 carboxylic groups. We assign the second component of the continuum band to the proton uptake complex, a cluster with an excess proton reminiscent to the PRC but located in the cytoplasmic domain and possibly stabilized by D38. Our findings refine the current interpretation of the continuum band and call for a reevaluation of the last proton transfer steps in bacteriorhodopsin.
Topics: Bacteriorhodopsins; Buffers; Cytoplasm; Hydrogen-Ion Concentration; Kinetics; Metabolic Networks and Pathways; Models, Molecular; Molecular Conformation; Protein Binding; Protons; Spectroscopy, Fourier Transform Infrared; Water
PubMed: 29203649
DOI: 10.1073/pnas.1707993114 -
Biochimica Et Biophysica Acta.... Jul 2022The proton pumping cycle of archaerhodopsin-2 (aR2) was investigated over a wide pH range and at different salt concentrations. We have found that two substates, which...
The proton pumping cycle of archaerhodopsin-2 (aR2) was investigated over a wide pH range and at different salt concentrations. We have found that two substates, which are spectroscopically and kinetically distinguishable, occur in the O intermediate. The first O-intermediate (O1) absorbs maximumly at ~580 nm, whereas the late O-intermediate (O2) absorbs maximumly at 605 nm. At neutral pH, O1 is in rapid equilibrium with the N intermediate. When the medium pH is increased, O1 becomes less stable than N and, in proportion to the amount of O1 in the dynamic equilibrium between N and O1, the formation rate of O2 decreases. By contrast, the decay rate of O2 increases ~100 folds when the pH of a low-salt membrane suspension is increased from 5.5 to 7.5 or when the salt concentration is increased to 2 M KCl. Together with our recent study on two substates in the O intermediate of bacteriorhodopsin (bR), the present study suggests that the thermally activated re-isomerization of the retinylidene chromophore into the initial all-trans configuration takes place in the O1-to-O2 transition; that is, O1 contains a distorted 13-cis chromophore. It is also found that the pKa value of the key ionizable residue (Asp101, Asp96) in the proton uptake channel is elevated in the O1 state of aR2 as compared to the O1 state of bR. This implies that the structural property of O1 in the aR2 photocycle can be investigated over a wide pH range.
Topics: Bacteriorhodopsins; Hydrogen-Ion Concentration; Light; Proton Pumps; Protons
PubMed: 35304864
DOI: 10.1016/j.bbamem.2022.183919 -
Biophysical Journal May 1995The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted conditions and can...
The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted conditions and can exist in chloride-free and chloride-binding forms. To describe the photochemical cycle of the all-trans, chloride-binding state that is associated with the transport, and thereby initiate study of the chloride translocation mechanism, one must first dissect the contributions of these species to the measured spectral changes. We resolved the multiple photochemical reactions by determining flash-induced difference spectra and photocycle kinetics in halorhodopsin-containing membranes prepared from Halobacterium salinarium, with light- and dark-adapted samples at various chloride concentrations. The high expression of cloned halorhodopsin made it possible to do these measurements with unfractionated cell envelope membranes in which the chromophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the flash-induced changes at selected wavelengths allowed separating the spectral changes into components and assigning them to the individual photocycles. According to the results, a substantial revision of the photocycle model for H. salinarium halorhodopsin, and its dependence on chloride, is required. The cycle of the all-trans chloride-binding form is described by the scheme, HR-hv-->K<==>L1<==>L2<==>N-->HR, where HR, K, L, and N designate halorhodopsin and its photointermediates. Unlike the earlier models, this is very similar to the photoreaction of bacteriorhodopsin when deprotonation of the Schiff base is prevented (e.g., at low pH or in the D85N mutant). Also unlike in the earlier models, no step in this photocycle was noticeably affected when the chloride concentration was varied between 20 mM and 2 M in an attempt to identify a chloride-binding reaction.
Topics: Bacteriorhodopsins; Halobacterium; Halorhodopsins; Kinetics; Light; Mathematics; Models, Theoretical; Retinaldehyde
PubMed: 7612849
DOI: 10.1016/S0006-3495(95)80385-1 -
Communications Biology Feb 2023The K intermediate of proton pumping bacteriorhodopsin is the first intermediate generated after isomerization of retinal to the 13-cis form. Although various structures...
The K intermediate of proton pumping bacteriorhodopsin is the first intermediate generated after isomerization of retinal to the 13-cis form. Although various structures have been reported for the K intermediate until now, these differ from each other, especially in terms of the conformation of the retinal chromophore and its interaction with surrounding residues. We report here an accurate X-ray crystallographic analysis of the K structure. The polyene chain of 13-cis retinal is observed to be S-shaped. The side chain of Lys216, which is covalently bound to retinal via the Schiff-base linkage, interacts with residues, Asp85 and Thr89. In addition, the Nζ-H of the protonated Schiff-base linkage interacts with a residue, Asp212 and a water molecule, W402. Based on quantum chemical calculations for this K structure, we examine the stabilizing factors of distorted conformation of retinal and propose a relaxation manner to the next L intermediate.
Topics: Bacteriorhodopsins; Models, Molecular; Proton Pumps; Molecular Conformation; Ion Transport
PubMed: 36808185
DOI: 10.1038/s42003-023-04554-2