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Biochemistry. Biokhimiia Oct 2023The diversity of the retinal-containing proteins (rhodopsins) in nature is extremely large. Fundamental similarity of the structure and photochemical properties unites... (Review)
Review
The diversity of the retinal-containing proteins (rhodopsins) in nature is extremely large. Fundamental similarity of the structure and photochemical properties unites them into one family. However, there is still a debate about the origin of retinal-containing proteins: divergent or convergent evolution? In this review, based on the results of our own and literature data, a comparative analysis of the similarities and differences in the photoconversion of the rhodopsin of types I and II is carried out. The results of experimental studies of the forward and reverse photoreactions of the bacteriorhodopsin (type I) and visual rhodopsin (type II) rhodopsins in the femto- and picosecond time scale, photo-reversible reaction of the octopus rhodopsin (type II), photovoltaic reactions, as well as quantum chemical calculations of the forward photoreactions of bacteriorhodopsin and visual rhodopsin are presented. The issue of probable convergent evolution of type I and type II rhodopsins is discussed.
Topics: Rhodopsin; Bacteriorhodopsins; Photochemistry
PubMed: 38105022
DOI: 10.1134/S0006297923100097 -
Biochemistry. Biokhimiia Oct 2023In the bioenergetics studies, the direct electrometric method played an important role. This method is based on measuring the electrical potential difference (Δψ)... (Review)
Review
In the bioenergetics studies, the direct electrometric method played an important role. This method is based on measuring the electrical potential difference (Δψ) between two compartments of the experimental cell generated by some membrane proteins. These proteins are incorporated into closed lipid-protein membrane vesicles associated with an artificial lipid membrane that separates the compartments. The very existence of such proteins able to generate Δψ was one of the consequences of Peter Mitchell's chemiosmotic concept. The discovery and investigation of their functioning contributed to the recognition of this concept and, eventually the well-deserved awarding of the Nobel Prize to P. Mitchell. Lel A. Drachev (1926-2022) was one of the main authors of the direct electrometrical method. With his participation, key studies were carried out on the electrogenesis of photosynthetic and respiratory membrane proteins, including bacteriorhodopsin, visual rhodopsin, photosynthetic bacterial reaction centers, cytochrome oxidase and others.
Topics: Photosynthetic Reaction Center Complex Proteins; Bacteria; Electron Transport Complex IV; Lipids
PubMed: 38105014
DOI: 10.1134/S0006297923100012 -
Advanced Science (Weinheim,... Apr 2024Controlling the pH at the microliter scale can be useful for applications in research, medicine, and industry, and therefore represents a valuable application for...
Controlling the pH at the microliter scale can be useful for applications in research, medicine, and industry, and therefore represents a valuable application for synthetic biology and microfluidics. The presented vesicular system translates light of different colors into specific pH changes in the surrounding solution. It works with the two light-driven proton pumps bacteriorhodopsin and blue light-absorbing proteorhodopsin Med12, that are oriented in opposite directions in the lipid membrane. A computer-controlled measuring device implements a feedback loop for automatic adjustment and maintenance of a selected pH value. A pH range spanning more than two units can be established, providing fine temporal and pH resolution. As an application example, a pH-sensitive enzyme reaction is presented where the light color controls the reaction progress. In summary, light color-controlled pH-adjustment using engineered proteoliposomes opens new possibilities to control processes at the microliter scale in different contexts, such as in synthetic biology applications.
Topics: Hydrogen-Ion Concentration; Bacteriorhodopsins; Proteolipids
PubMed: 38342618
DOI: 10.1002/advs.202307524 -
Nature Communications Mar 2024Discovered over 50 years ago, bacteriorhodopsin is the first recognized and most widely studied microbial retinal protein. Serving as a light-activated proton pump, it...
Discovered over 50 years ago, bacteriorhodopsin is the first recognized and most widely studied microbial retinal protein. Serving as a light-activated proton pump, it represents the archetypal ion-pumping system. Here we compare the photochemical dynamics of bacteriorhodopsin light and dark-adapted forms with that of the first metastable photocycle intermediate known as "K". We observe that following thermal double isomerization of retinal in the dark from bio-active all-trans 15-anti to 13-cis, 15-syn, photochemistry proceeds even faster than the ~0.5 ps decay of the former, exhibiting ballistic wave packet curve crossing to the ground state. In contrast, photoexcitation of K containing a 13-cis, 15-anti chromophore leads to markedly multi-exponential excited state decay including much slower stages. QM/MM calculations, aimed to interpret these results, highlight the crucial role of protonation, showing that the classic quadrupole counterion model poorly reproduces spectral data and dynamics. Single protonation of ASP212 rectifies discrepancies and predicts triple ground state structural heterogeneity aligning with experimental observations. These findings prompt a reevaluation of counter ion protonation in bacteriorhodopsin and contribute to the broader understanding of its photochemical dynamics.
Topics: Bacteriorhodopsins; Photochemistry; Proton Pumps; Light
PubMed: 38459010
DOI: 10.1038/s41467-024-46061-w -
Biophysical Journal Nov 2023The experimentally measured stretching vibrational frequencies of O-D [ν(donor)] and C=O [ν(donor)] H-bond donor groups can provide valuable information about the...
The experimentally measured stretching vibrational frequencies of O-D [ν(donor)] and C=O [ν(donor)] H-bond donor groups can provide valuable information about the H-bonds in proteins. Here, using a quantum mechanical/molecular mechanical approach, the relationship between these vibrational frequencies and the difference in pK values between H-bond donor and acceptor groups [ΔpK(donor … acceptor)] in bacteriorhodopsin and photoactive yellow protein environments was investigated. The results show that ν(donor) is correlated with ΔpK(donor … acceptor), regardless of the specific protein environment. ν(donor) is also correlated with ΔpK(donor … acceptor), although the correlation is weak because the C=O bond does not have a proton. Importantly, the shifts in ν(donor) and ν(donor) are not caused by changes in pK(donor) alone, but rather by changes in ΔpK(donor … acceptor). Specifically, a decrease in ΔpK(donor … acceptor) can lead to proton release from the H-bond donor group toward the acceptor group, resulting in shifts in the vibrational frequencies of the protein environment. These findings suggest that changes in the stretching vibrational frequencies, in particular ν(donor), can be used to monitor proton transfer in protein environments.
Topics: Protons; Proteins; Vibration
PubMed: 37838831
DOI: 10.1016/j.bpj.2023.10.012 -
Journal of Molecular Biology Mar 2024Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is the first discovered natural light-gated ion channel that shows higher selectivity to K than to Na...
Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is the first discovered natural light-gated ion channel that shows higher selectivity to K than to Na and therefore is used to silence neurons with light (optogenetics). Replacement of the conserved cysteine residue in the transmembrane helix 3 (Cys110) with alanine or threonine results in a >1,000-fold decrease in the channel closing rate. The phenotype of the corresponding mutants in channelrhodopsin 2 is attributed to breaking of a specific interhelical hydrogen bond (the "DC gate"). Unlike CrChR2 and other ChRs with long distance "DC gates", the HcKCR1 structure does not reveal any hydrogen bonding partners to Cys110, indicating that the mutant phenotype is likely caused by disruption of direct interaction between this residue and the chromophore. In HcKCR1_C110A, fast photochemical conversions corresponding to channel gating were followed by dramatically slower absorption changes. Full recovery of the unphotolyzed state in HcKCR1_C110A was extremely slow with two time constants 5.2 and 70 min. Analysis of the light-minus-dark difference spectra during these slow processes revealed accumulation of at least four spectrally distinct blue light-absorbing photocycle intermediates, L, M and M, and a UV light-absorbing form, typical of bacteriorhodopsin-like channelrhodopsins from cryptophytes. Our results contribute to better understanding of the mechanistic links between the chromophore photochemistry and channel conductance, and provide the basis for using HcKCR1_C110A as an optogenetic tool.
Topics: Channelrhodopsins; Light; Rhinosporidium; Ion Channel Gating; Mutation; Cysteine; Protein Conformation, alpha-Helical; Humans; HEK293 Cells; Optogenetics; Conserved Sequence; Amino Acid Substitution
PubMed: 37802216
DOI: 10.1016/j.jmb.2023.168298 -
Biosensors Oct 2023An effective early diagnosis is important for rheumatoid arthritis (RA) management. This study reveals a novel RA detection method using bacteriorhodopsin as a...
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) 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.
Topics: Humans; Bacteriorhodopsins; Gold; Metal Nanoparticles; Arthritis, Rheumatoid; Peptides; Enzyme-Linked Immunosorbent Assay; Biosensing Techniques
PubMed: 37887122
DOI: 10.3390/bios13100929 -
Structural Dynamics (Melville, N.Y.) May 2023Low-pass spectral analysis (LPSA) is a recently developed dynamics retrieval algorithm showing excellent retrieval properties when applied to model data affected by...
Low-pass spectral analysis (LPSA) is a recently developed dynamics retrieval algorithm showing excellent retrieval properties when applied to model data affected by extreme incompleteness and stochastic weighting. In this work, we apply LPSA to an experimental time-resolved serial femtosecond crystallography (TR-SFX) dataset from the membrane protein bacteriorhodopsin (bR) and analyze its parametric sensitivity. While most dynamical modes are contaminated by nonphysical high-frequency features, we identify two dominant modes, which are little affected by spurious frequencies. The dynamics retrieved using these modes shows an isomerization signal compatible with previous findings. We employ synthetic data with increasing timing uncertainty, increasing incompleteness level, pixel-dependent incompleteness, and photon counting errors to investigate the root cause of the high-frequency contamination of our TR-SFX modes. By testing a range of methods, we show that timing errors comparable to the dynamical periods to be retrieved produce a smearing of dynamical features, hampering dynamics retrieval, but with no introduction of spurious components in the solution, when convergence criteria are met. Using model data, we are able to attribute the high-frequency contamination of low-order dynamical modes to the high levels of noise present in the data. Finally, we propose a method to handle missing observations that produces a substantial dynamics retrieval improvement from synthetic data with a significant static component. Reprocessing of the bR TR-SFX data using the improved method yields dynamical movies with strong isomerization signals compatible with previous findings.
PubMed: 37275629
DOI: 10.1063/4.0000178 -
Biochemistry. Biokhimiia Oct 2023Retinal-containing light-sensitive proteins - rhodopsins - are found in many microorganisms. Interest in them is largely explained by their role in light energy storage... (Review)
Review
Retinal-containing light-sensitive proteins - rhodopsins - are found in many microorganisms. Interest in them is largely explained by their role in light energy storage and photoregulation in microorganisms, as well as the prospects for their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, the retinal protein of Exiguobacterium sibiricum. What distinguishes ESR from homologous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with the hydrogen bond of the proton acceptor Asp85 with the His57 residue, determines functional characteristics of ESR as a proton pump. This review examines the results of ESR studies conducted using various methods, including direct electrometry. Comparison of the obtained data with the results of structural studies and with other retinal proteins allows us to draw conclusions about the mechanisms of transport of hydrogen ions in ESR and similar retinal proteins.
Topics: Protons; Ion Transport; Proton Pumps; Rhodopsins, Microbial; Bacteriorhodopsins
PubMed: 38105023
DOI: 10.1134/S0006297923100103 -
Structural Dynamics (Melville, N.Y.) Mar 2024Understanding the chemical reactions that give rise to functional biological systems is at the core of structural biology. As techniques are developed to study the...
Understanding the chemical reactions that give rise to functional biological systems is at the core of structural biology. As techniques are developed to study the chemical reactions that drive biological processes, it must be ensured that the reaction occurring is indeed a biologically relevant pathway. There is mounting evidence indicating that there has been a propagation of systematic error in the study of photoactive biological processes; the optical methods used to probe the structural dynamics of light activated protein functions have failed to ensure that the photoexcitation prepares a well-defined initial state relevant to the biological process of interest. Photoexcitation in nature occurs in the linear (one-photon per chromophore) regime; however, the extreme excitation conditions used experimentally give rise to biologically irrelevant multiphoton absorption. To evaluate and ensure the biological relevance of past and future experiments, a theoretical framework has been developed to determine the excitation conditions, which lead to resonant multiphoton absorption (RMPA) and thus define the excitation limit in general for the study of structural dynamics within the 1-photon excitation regime. Here, we apply the theoretical model to bacteriorhodopsin (bR) and show that RMPA occurs when excitation conditions exceed the linear saturation threshold, well below typical excitation conditions used in this class of experiments. This work provides the guidelines to ensure excitation in the linear 1-photon regime is relevant to biological and chemical processes.
PubMed: 38433875
DOI: 10.1063/4.0000239