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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 -
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 -
Biophysics and Physicobiology Mar 2023It marked half a century since the discovery of bacteriorhodopsin two years ago. On this occasion, I have revisited historically important diffraction studies of this...
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.
PubMed: 38362329
DOI: 10.2142/biophysico.bppb-v20.s006 -
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 -
Acta Crystallographica. Section D,... Feb 2024Over the last decade, the development of time-resolved serial crystallography (TR-SX) at X-ray free-electron lasers (XFELs) and synchrotrons has allowed researchers to...
Over the last decade, the development of time-resolved serial crystallography (TR-SX) at X-ray free-electron lasers (XFELs) and synchrotrons has allowed researchers to study phenomena occurring in proteins on the femtosecond-to-minute timescale, taking advantage of many technical and methodological breakthroughs. Protein crystals of various sizes are presented to the X-ray beam in either a static or a moving medium. Photoactive proteins were naturally the initial systems to be studied in TR-SX experiments using pump-probe schemes, where the pump is a pulse of visible light. Other reaction initiations through small-molecule diffusion are gaining momentum. Here, selected examples of XFEL and synchrotron time-resolved crystallography studies will be used to highlight the specificities of the various instruments and methods with respect to time resolution, and are compared with cryo-trapping studies.
Topics: Synchrotrons; Crystallography; Crystallography, X-Ray; X-Rays; Proteins; Lasers
PubMed: 38265875
DOI: 10.1107/S2059798323011002 -
Gold nanoparticle-powered screening of membrane protein-specific lipids from complex lipid mixtures.Analytical Biochemistry Apr 2024Membrane proteins (MPs) are affected by binding of specific lipids. We previously developed a methodology for systematically analyzing MP-lipid interactions leveraging...
Membrane proteins (MPs) are affected by binding of specific lipids. We previously developed a methodology for systematically analyzing MP-lipid interactions leveraging surface plasmon resonance (SPR). In this method, the gold sensor chip surface was modified with a self-assembled monolayer (SAM), which allowed for a larger amount of MP-immobilization. However, the laborious lipid purification step remained a bottleneck. To address this issue, a new strategy has been developed utilizing gold nanoparticles (AuNPs) instead of the gold sensor chip. AuNPs were coated with SAM, on which MP was covalently anchored. The MP-immobilized AuNPs were mixed with a lipid mixture, and the recovered lipids were quantified by LC-MS. Bacteriorhodopsin (bR) was used as an MP to demonstrate this concept. We optimized immobilization conditions and confirmed the efficient immobilization of bR by dynamic light scattering and electron micrographs. Washing conditions for pulldown experiments were optimized to efficiently remove non-specific lipids. A new binding index was introduced to qualitatively reproduce the known affinity of lipids for bR. Consequently, the low-abundant and least-studied lipid S-TeGD was identified as a candidate for bR-specific lipids. This technique can skip the laborious lipid purification process, accelerating the screening of MP-specific lipids from complex lipid mixtures.
Topics: Membrane Lipids; Gold; Membrane Proteins; Metal Nanoparticles; Surface Plasmon Resonance
PubMed: 38141800
DOI: 10.1016/j.ab.2023.115447 -
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 -
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 -
Acta Crystallographica. Section D,... Jan 2024The technique of time-resolved macromolecular crystallography (TR-MX) has recently been rejuvenated at synchrotrons, resulting in the design of dedicated beamlines....
The technique of time-resolved macromolecular crystallography (TR-MX) has recently been rejuvenated at synchrotrons, resulting in the design of dedicated beamlines. Using pump-probe schemes, this should make the mechanistic study of photoactive proteins and other suitable systems possible with time resolutions down to microseconds. In order to identify relevant time delays, time-resolved spectroscopic experiments directly performed on protein crystals are often desirable. To this end, an instrument has been built at the icOS Lab (in crystallo Optical Spectroscopy Laboratory) at the European Synchrotron Radiation Facility using reflective focusing objectives with a tuneable nanosecond laser as a pump and a microsecond xenon flash lamp as a probe, called the TR-icOS (time-resolved icOS) setup. Using this instrument, pump-probe spectra can rapidly be recorded from single crystals with time delays ranging from a few microseconds to seconds and beyond. This can be repeated at various laser pulse energies to track the potential presence of artefacts arising from two-photon absorption, which amounts to a power titration of a photoreaction. This approach has been applied to monitor the rise and decay of the M state in the photocycle of crystallized bacteriorhodopsin and showed that the photocycle is increasingly altered with laser pulses of peak fluence greater than 100 mJ cm, providing experimental laser and delay parameters for a successful TR-MX experiment.
Topics: Spectrum Analysis; Proteins; Synchrotrons; Crystallography; Light
PubMed: 38088897
DOI: 10.1107/S2059798323010483