نتائج البحث - "Bacteriorhodopsins"

1

Cancer cells targeting with genetically engineered constructs based on a pH-dependent membrane insertion peptide and fluorescent protein

المؤلفون: Anastasiya Yu. Frolova, Alexey A. Pakhomov, Dmitry L. Kakuev, Anna S. Sungurova, Sergey M. Deyev, Vladimir I. Martynov

المصدر: Biochemical and Biophysical Research Communications. 612:141-146

مصطلحات موضوعية: Bacteriorhodopsins, Neoplasms, Cell Membrane, Biophysics, Humans, Cell Biology, Hydrogen-Ion Concentration, Peptides, Molecular Biology, Biochemistry, Fluorescence, HeLa Cells

الوصف: The targeted delivery of nanodrugs to malignant neoplasm is one of the most pressing challenges in the development of modern medicine. It was reported earlier that a bacteriorhodopsin-derived pH low insertion peptide (pHLIP) targets acidic tumors and has the ability to translocate low molecular weight cargoes across the cancer cell membrane. Here, to better understand the potential of pHLIP-related technologies, we used genetically engineered fluorescent protein (EGFP) as a model protein cargo and examined targeting efficiencies of EGFP-pHLIP hybrid constructs in vitro with the HeLa cell line at different pHs. By two independent monitoring methods we observed an increased binding affinity of EGFP-pHLIP fusions to HeLa cells at pH below 6.8. Confocal images of EGFP-pHLIP-treated cells showed bright fluorescence associated with the cell membrane and fluorescent dots localized inside the cell, that became brighter with time. To elucidate the pHLIP-mediated EGFP cell entry mechanisms, we performed a series of experiments with specific inhibitors of endocytosis. Our results imply that EGFP-pHLIP internalization is realized by endocytosis of various types.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9f425a2b5a2c8ca9de7d1dc60ff17b6cTest
https://doi.org/10.1016/j.bbrc.2022.04.112Test

2

Single molecule kinetics of bacteriorhodopsin by HS-AFM

المؤلفون: Perrino, Alma P., Miyagi, Atsushi, Scheuring, Simon

المصدر: Nature Communications, Vol 12, Iss 1, Pp 1-10 (2021)
Nature Communications

الوصف: Bacteriorhodopsin is a seven-helix light-driven proton-pump that was structurally and functionally extensively studied. Despite a wealth of data, the single molecule kinetics of the reaction cycle remain unknown. Here, we use high-speed atomic force microscopy methods to characterize the single molecule kinetics of wild-type bR exposed to continuous light and short pulses. Monitoring bR conformational changes with millisecond temporal resolution, we determine that the cytoplasmic gate opens 2.9 ms after photon absorption, and stays open for proton capture for 13.2 ms. Surprisingly, a previously active protomer cannot be reactivated for another 37.6 ms, even under excess continuous light, giving a single molecule reaction cycle of ~20 s−1. The reaction cycle slows at low light where the closed state is prolonged, and at basic or acidic pH where the open state is extended.
Here, the authors use high-speed atomic force microscopy (HS-AFM) methods to characterize the single molecule kinetics of wild-type bacteriorhodopsin (bR) with millisecond temporal resolution, providing new insights into the bR conformational cycle.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::318367eb2cc3e7ccd6a60c6cffaffb3eTest
https://doaj.org/article/8cb3eaef9fad4202a1ed7133ccef787eTest

3

Two substates in the O intermediate of the light-driven proton pump archaerhodopsin-2

المؤلفون: Tsutomu Kouyama, Kunio Ihara

المصدر: Biochimica et biophysica acta. Biomembranes. 1864(7)

مصطلحات موضوعية: Light, Bacteriorhodopsins, Biophysics, Cell Biology, Hydrogen-Ion Concentration, Proton Pumps, Protons, Biochemistry

الوصف: 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

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::3b1282e37ae08df4c4193101e4d8d477Test
https://pubmed.ncbi.nlm.nih.gov/35304864Test

4

Proton transfer reactions in donor site mutants of ESR, a retinal protein from Exiguobacterium sibiricum

المؤلفون: Lada E, Petrovskaya, Evgeniy P, Lukashev, Sergey A, Siletsky, Eleonora S, Imasheva, Jennifer M, Wang, Mahir D, Mamedov, Elena A, Kryukova, Dmitriy A, Dolgikh, Andrei B, Rubin, Mikhail P, Kirpichnikov, Sergei P, Balashov, Janos K, Lanyi

المصدر: Journal of Photochemistry and Photobiology B: Biology. 234:112529

مصطلحات موضوعية: Radiation, Exiguobacterium, Radiological and Ultrasound Technology, Bacteriorhodopsins, Biophysics, Radiology, Nuclear Medicine and imaging, Hydrogen-Ion Concentration, Proton Pumps, Protons, Schiff Bases

الوصف: Light-driven proton transport by microbial retinal proteins such as archaeal bacteriorhodopsin involves carboxylic residues as internal proton donors to the catalytic center which is a retinal Schiff base (SB). The proton donor, Asp96 in bacteriorhodopsin, supplies a proton to the transiently deprotonated Schiff base during the photochemical cycle. Subsequent proton uptake resets the protonated state of the donor. This two step process became a distinctive signature of retinal based proton pumps. Similar steps are observed also in many natural variants of bacterial proteorhodopsins and xanthorhodopsins where glutamic acid residues serve as a proton donor. Recently, however, an exception to this rule was found. A retinal protein from Exiguobacterium sibiricum, ESR, contains a Lys residue in place of Asp or Glu, which facilitates proton transfer from the bulk to the SB. Lys96 can be functionally replaced with the more common donor residues, Asp or Glu. Proton transfer to the SB in the mutants containing these replacements (K96E and K96D/A47T) is much faster than in the proteins lacking the proton donor (K96A and similar mutants), and in the case of K96D/A47T, comparable with that in the wild type, indicating that carboxylic residues can replace Lys96 as proton donors in ESR. We show here that there are important differences in the functioning of these residues in ESR from the way Asp96 functions in bacteriorhodopsin. Reprotonation of the SB and proton uptake from the bulk occur almost simultaneously during the M to N transition (as in the wild type ESR at neutral pH), whereas in bacteriorhodopsin these two steps are well separated in time and occur during the M to N and N to O transitions, respectively.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::c710818a78cbc8ccdfc982def69e4532Test
https://doi.org/10.1016/j.jphotobiol.2022.112529Test

5

Amino acid deprotonation rates from classical force fields

المؤلفون: Themis Lazaridis, Aliasghar Sepehri

المصدر: The Journal of Chemical Physics. 157:085101

مصطلحات موضوعية: Aspartic Acid, Kinetics, Bacteriorhodopsins, General Physics and Astronomy, Amino Acids, Hydrogen-Ion Concentration, Protons, Physical and Theoretical Chemistry

الوصف: Acid ionization constants (pKa’s) of titratable amino acid side chains have received a large amount of experimental and theoretical attention. In many situations, however, the rates of protonation and deprotonation, kon and koff, may also be important, for example, in understanding the mechanism of action of proton channels or membrane proteins that couple proton transport to other processes. Protonation and deprotonation involve the making and breaking of covalent bonds, which cannot be studied by classical force fields. However, environment effects on the rates should be captured by such methods. Here, we present an approach for estimating deprotonation rates based on Warshel’s extension of Marcus’s theory of electron transfer, with input from molecular simulations. The missing bond dissociation energy is represented by a constant term determined by fitting the pKa value in solution. The statistics of the energy gap between protonated and deprotonated states is used to compute free energy curves of the two states and, thus, free energy barriers, from which the rate can be deduced. The method is applied to Glu, Asp, and His in bulk solution and select membrane proteins: the M2 proton channel, bacteriorhodopsin, and cytochrome c oxidase.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9b0d4c7aa0fb4fc289ecab00245679a2Test
https://doi.org/10.1063/5.0101960Test

6

Lokiarchaeota archaeon schizorhodopsin-2 (LaSzR2) is an inward proton pump displaying a characteristic feature of acid-induced spectral blue-shift

المؤلفون: Yuki Sudo, Keiichi Kojima, Takashi Kikukawa, Masumi Hasegawa, Susumu Yoshizawa, Masaki Nakama, Marie Kurihara

المصدر: Scientific Reports

الوصف: The photoreactive protein rhodopsin is widespread in microorganisms and has a variety of photobiological functions. Recently, a novel phylogenetically distinctive group named ‘schizorhodopsin (SzR)’ has been identified as an inward proton pump. We performed functional and spectroscopic studies on an uncharacterised schizorhodopsin from the phylum Lokiarchaeota archaeon. The protein, LaSzR2, having an all-trans-retinal chromophore, showed inward proton pump activity with an absorption maximum at 549 nm. The pH titration experiments revealed that the protonated Schiff base of the retinal chromophore (Lys188, pKa = 12.3) is stabilised by the deprotonated counterion (presumably Asp184, pKa = 3.7). The flash-photolysis experiments revealed the presence of two photointermediates, K and M. A proton was released and uptaken from bulk solution upon the formation and decay of the M intermediate. During the M-decay, the Schiff base was reprotonated by the proton from a proton donating residue (presumably Asp172). These properties were compared with other inward (SzRs and xenorhodopsins, XeRs) and outward proton pumps. Notably, LaSzR2 showed acid-induced spectral ‘blue-shift’ due to the protonation of the counterion, whereas outward proton pumps showed opposite shifts (red-shifts). Thus, we can distinguish between inward and outward proton pumps by the direction of the acid-induced spectral shift.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::6ce5482258ccb5df24dd63a38f8ff15cTest
https://doi.org/10.1038/s41598-020-77936-9Test

7

Collective exchange processes reveal an active site proton cage in bacteriorhodopsin

المؤلفون: Roland R. Netz, Peter Hegemann, Andrew J. Nieuwkoop, Florian N. Brünig, Daniel Friedrich, Hartmut Oschkinat

المصدر: Communications Biology
Communications Biology, Vol 3, Iss 1, Pp 1-9 (2020)

مصطلحات موضوعية: 0301 basic medicine, Models, Molecular, Magnetic Resonance Spectroscopy, genetic structures, Proton, Protein Conformation, Nuclear Theory, Biophysics, Medicine (miscellaneous), Protonation, Molecular Dynamics Simulation, 010402 general chemistry, 01 natural sciences, Biochemistry, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Catalytic Domain, Magic angle spinning, Nuclear Experiment, lcsh:QH301-705.5, biology, Chemistry, Hydrogen bond, 500 Naturwissenschaften und Mathematik::530 Physik::530 Physik, Temperature, Active site, Bacteriorhodopsin, Hydrogen Bonding, Hydrogen-Ion Concentration, Tautomer, Chemical biology, 0104 chemical sciences, 030104 developmental biology, lcsh:Biology (General), Chemical physics, Bacteriorhodopsins, biology.protein, Physics::Accelerator Physics, Protons, General Agricultural and Biological Sciences, Structural biology, Isomerization

الوصف: Proton translocation across membranes is vital to all kingdoms of life. Mechanistically, it relies on characteristic proton flows and modifications of hydrogen bonding patterns, termed protonation dynamics, which can be directly observed by fast magic angle spinning (MAS) NMR. Here, we demonstrate that reversible proton displacement in the active site of bacteriorhodopsin already takes place in its equilibrated dark-state, providing new information on the underlying hydrogen exchange processes. In particular, MAS NMR reveals proton exchange at D85 and the retinal Schiff base, suggesting a tautomeric equilibrium and thus partial ionization of D85. We provide evidence for a proton cage and detect a preformed proton path between D85 and the proton shuttle R82. The protons at D96 and D85 exchange with water, in line with ab initio molecular dynamics simulations. We propose that retinal isomerization makes the observed proton exchange processes irreversible and delivers a proton towards the extracellular release site.
Daniel Friedrich et al. show that reversible proton translocation occurs in the dark–state of bacteriorhodopsin, involving the retinal Schiff base and D85 exchanging protons with H2O. They find evidence of an active site proton cage and possible proton transfer via R82.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b648e6d6d17185e60570e432b035b46aTest
https://pubmed.ncbi.nlm.nih.gov/31925324Test

8

pH-dependent thermodynamic intermediates of pHLIP membrane insertion determined by solid-state NMR spectroscopy

المؤلفون: Anqi Zhang, Samuel Z. Hanz, Lukas M. Klees, Bianca Chakravorty, Sarah A. Otieno, Wei Qiang, Ming An

المصدر: Proceedings of the National Academy of Sciences of the United States of America. 115(48)

الوصف: The applications of the pH low insertion peptide (pHLIP) in cancer diagnosis and cross-membrane cargo delivery have drawn increasing attention in the past decade. With its origin as the transmembrane (TM) helix C of bacteriorhodopsin, pHLIP is also an important model for understanding how pH can affect the folding and topogenesis of a TM α-helix. Protonations of multiple D/E residues transform pHLIP from an unstructured coil at membrane surface (known as state II, at pH ≥ 7) to a TM α-helix (state III, pH ≤ 5.3). While these initial and end states of pHLIP insertion have been firmly established, what happens at the intervening pH values is less clear. However, the intervening pH range is most relevant to pHLIP-cell interactions in the acidic extracellular tumor environment (and in the endosomes within cells). Here, using advanced solid-state NMR spectroscopy with palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine unilamellar vesicles as the model membrane, we systematically examined the state of pHLIP-membrane interactions (in terms of the membrane locations of D/E residues, as well as lipid dynamics) at the intervening pH values of 6.4, 6.1, and 5.8, along with the known states at pH 7.4 and 5.3. Thermodynamic intermediate states distinct from the initial and end states were discovered to exist at each of the intervening pH examined. They support a multistage model of pHLIP insertion in which the D/E titrations occur in a defined sequence at distinct intermediate pH values. This multistage model has important ramifications in pHLIP applications.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::8772cff11a68c7e10da53b9c47f8076bTest
https://pubmed.ncbi.nlm.nih.gov/30442664Test

9

Biological Optical-to-Chemical Signal Conversion Interface: A Small-Scale Modulator for Molecular Communications

المؤلفون: Vahid Jamali, Arman Ahmadzadeh, Andreas Burkovski, Jens Kirchner, Florian Irnstorfer, Robert Schober, Laura Grebenstein, Georg Fischer, Wayan Wicke, Renata Stavracakis Peixoto, Wiebke Zimmermann, Robert Weigel

المصدر: IEEE transactions on nanobioscience. 18(1)

مصطلحات موضوعية: FOS: Computer and information sciences, Interface (computing), Biomedical Engineering, Pharmaceutical Science, Medicine (miscellaneous), Computer Science - Emerging Technologies, Bioengineering, 02 engineering and technology, Signal, Models, Biological, Computers, Molecular, Escherichia coli, Electrical and Electronic Engineering, Microscale chemistry, Physics, Molecular communication, Detector, Estimator, Signal Processing, Computer-Assisted, Hydrogen-Ion Concentration, Proton Pumps, 021001 nanoscience & nanotechnology, Computer Science Applications, Emerging Technologies (cs.ET), Models, Chemical, Bacteriorhodopsins, Parametric model, 0210 nano-technology, Biological system, Biotechnology, Communication channel, Signal Transduction

الوصف: Although many exciting applications of molecular communication (MC) systems are envisioned to be at microscale, the MC testbeds reported so far are mostly at macroscale. To link the macroworld to the microworld, we propose and demonstrate a biological signal conversion interface that can also be seen as a microscale modulator. In particular, the proposed interface transduces an optical signal, which is controlled using an LED, into a chemical signal by changing the pH of the environment. The modulator is realized using E. coli bacteria as microscale entity expressing the light-driven proton pump gloeorhodopsin from Gloeobacter violaceus. Upon inducing external light stimuli, these bacteria locally change their surrounding pH level by exporting protons into the environment. To verify the effectiveness of the proposed optical-to-chemical signal converter, we analyze the pH signal measured by a pH sensor, which serves as receiver. We develop an analytical parametric model for the induced chemical signal as a function of the applied optical signal. Using this model, we derive a training-based channel estimator which estimates the parameters of the proposed model to fit the measurement data. We further derive the optimal maximum likelihood detector and a suboptimal low-complexity detector to recover the transmitted data from the measured received signal. It is shown that the proposed parametric model is in good agreement with the measurement data. Moreover, for an example scenario, we show that the proposed setup is able to successfully convert an optical signal representing a sequence of binary symbols into a chemical signal with a bit rate of 1 bit/minute and recover the transmitted data from the chemical signal using the proposed estimation and detection~schemes. The proposed modulator may form the basis for future MC testbeds and applications at microscale.
Comment: 28 pages, 10 figures. This work has been submitted to the IEEE for possible publication. Copyright may be transferred without notice, after which this version may no longer be accessible. arXiv admin note: substantial text overlap with arXiv:1804.05555

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::fdf41299884b3ec4bc2daaaa1d019a00Test
https://pubmed.ncbi.nlm.nih.gov/30235144Test

10

His57 controls the efficiency of ESR, a light-driven proton pump from Exiguobacterium sibiricum at low and high pH

المؤلفون: E. P. Lukashev, Andrei B. Rubin, D. A. Dolgikh, Lada E. Petrovskaya, Sergey A. Siletsky, Sergei P. Balashov, Mikhail P. Kirpichnikov, Vitaliy B. Borisov, Mahir D. Mamedov

المصدر: Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1862:148328

الوصف: ESR, a light-driven proton pump from Exiguobacterium sibiricum, contains a lysine residue (Lys96) in the proton donor site. Substitution of Lys96 with a nonionizable residue greatly slows reprotonation of the retinal Schiff base. The recent study of electrogenicity of the K96A mutant revealed that overall efficiency of proton transport is decreased in the mutant due to back reactions (Siletsky et al., BBA, 2019). Similar to members of the proteorhodopsin and xanthorhodopsin families, in ESR the primary proton acceptor from the Schiff base, Asp85, closely interacts with His57. To examine the role of His57 in the efficiency of proton translocation by ESR, we studied the effects of H57N and H57N/K96A mutations on the pH dependence of light-induced pH changes in suspensions of Escherichia coli cells, kinetics of absorption changes and electrogenic proton transfer reactions during the photocycle. We found that at low pH (

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::89761fd0a41d024df3c7ef9028794b9eTest
https://doi.org/10.1016/j.bbabio.2020.148328Test

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