A New Group of Eubacterial Light-Driven Proton Pumps Lacking the Carboxylic Proton Donor

AbstractThe green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.

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A new group of eubacterial light-driven retinal-binding proton pumps with an unusual cytoplasmic proton donor

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/j.bbabio.2015.08.003 ◽

2015 ◽

Vol 1847 (12) ◽

pp. 1518-1529 ◽

Cited By ~ 20

Author(s):

Andrew Harris ◽

Milena Ljumovic ◽

Ana-Nicoleta Bondar ◽

Yohei Shibata ◽

Shota Ito ◽

...

Keyword(s):

Proton Donor ◽

Proton Pumps

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Conductance Mechanisms of Rapidly Desensitizing Cation Channelrhodopsins from Cryptophyte Algae

mBio ◽

10.1128/mbio.00657-20 ◽

2020 ◽

Vol 11 (2) ◽

Author(s):

Oleg A. Sineshchekov ◽

Elena G. Govorunova ◽

Hai Li ◽

Yumei Wang ◽

Michael Melkonian ◽

...

Keyword(s):

Green Algae ◽

Molecular Mechanisms ◽

Flash Photolysis ◽

Continuous Light ◽

Proton Donor ◽

Continuous Illumination ◽

Proton Pumps ◽

Cation Conductance ◽

Vis Spectroscopy ◽

Rapid Accumulation

ABSTRACT Channelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. “Bacteriorhodopsin-like” cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, the sequences of BCCR match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here, we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption, Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function. IMPORTANCE Cation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-visible (UV-vis) spectroscopy and photoelectrophysiology reveal that the desensitization is caused by rapid accumulation of a nonconductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

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Conductance mechanisms of rapidly desensitizing cation channelrhodopsins from cryptophyte algae

10.1101/2020.03.20.001099 ◽

2020 ◽

Author(s):

Oleg A. Sineshchekov ◽

Elena G. Govorunova ◽

Hai Li ◽

Yumei Wang ◽

Michael Melkonian ◽

...

Keyword(s):

Green Algae ◽

Molecular Mechanisms ◽

Flash Photolysis ◽

Continuous Light ◽

Proton Donor ◽

Continuous Illumination ◽

Proton Pumps ◽

Cation Conductance ◽

Vis Spectroscopy ◽

Rapid Accumulation

ABSTRACTChannelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. “Bacteriorhodopsin-like” cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, BCCR sequences match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs, which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption and Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function.IMPORTANCECation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-vis spectroscopy and photoelectrophysiology reveal the desensitization is caused by rapid accumulation of a non-conductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

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Mechanistic Study and Development of Catalytic Reactions of Sm(II)

10.26434/chemrxiv.7355069 ◽

2018 ◽

Author(s):

Sandepan Maity ◽

Robert Flowers

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Proton Donor ◽

Catalytic Reactions ◽

Mechanistic Study ◽

Mechanistic Studies ◽

Cyclization Reactions ◽

Mechanistic Approach ◽

Donor Source

Despite the broad utility and application of SmI2in synthesis, the reagent is used in stoichiometric amounts and has a high molecular weight, resulting in a large amount of material being used for reactions requiring one or more equivalents of electrons. We report mechanistic studies on catalytic reactions of Sm(II) employing a terminal magnesium reductant and trimethyl silyl chloride in concert with a non-coordinating proton donor source. Reactions using this approach permitted reductions with as little as 1 mol% Sm. The mechanistic approach enabled catalysis employing HMPA as a ligand, facilitating the development of catalytic Sm(II) 5-exo-trig ketyl olefin cyclization reactions.

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In vitro antibacterial and antibiotic-potentiation activities of five edible plant extracts and mode of action against several MDR Gram-negative phenotypes

Investigational Medicinal Chemistry and Pharmacology ◽

10.31183/imcp.2020.00049 ◽

2020 ◽

Vol 4 (1) ◽

pp. 1-14

Author(s):

Carine M.N. Ngaffo ◽

Simplice B. Tankeo ◽

Michel-Gael F. Guefack ◽

Brice E. N. Wamba ◽

Paul Nayim ◽

...

Keyword(s):

Secondary Metabolites ◽

Bioactive Compounds ◽

Plant Extracts ◽

Theobroma Cacao ◽

Efflux Pumps ◽

Phoenix Dactylifera ◽

Food Plants ◽

Proton Pumps ◽

Gram Negative ◽

E Coli

Abstract Background: Bacterial infections involving the multidrug resistant (MDR) strains are among the top leading causes of death throughout the world. Healthcare system across the globe has been suffering from an extra-ordinary burden in terms of looking for the new and more potent antimicrobial compounds. The aim of the present study was to determine the antibacterial activity of some Cameroonian edible plants (Garcinia lucida bark, Phoenix dactylifera pericarps, Theobroma cacao pod, Solanum macrocarpon leaves and Termitomyces titanicus whole plant) and their antibiotics-potentiation effects against some MDR Gram-negative bacteria phenotypes expressing efflux pumps (Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa and Providencia stuartii strains). Methods: The antibacterial activities of plant extract alone and in combination with usual antibiotics were carried out using the micro-dilution method. The effects of the most active plant extract (Garcinia lucida bark) on H+-ATPase-mediated proton pumps and on bacterial growth kinetic were performed using experimental protocols, while qualitative reference methods were used to highligh the major groups of secondary metabolites present in the extracts. Results: Qualitative phytochemical screening of plant extracts indicated that all analysed secondary metabolites were present in Theobroma cacao and Termitomyces titanicus while one (saponins) of them was absent in Garcinia lucida and Solanum macrocarpon. Only three of them (polyphenols, flavonoids and saponins) were detected in Phoenix dactylifera. Antibacterial essays showed that G. lucida was the most active plant as it inhibited the growth of all studied bacteria with strong activity (MIC<100 µg/mL) against E. coli ATCC8739, significant activity (100≤MIC≤512 µg/mL) against 80% of bacteria and moderate activity (512<MIC≤2048 µg/mL) against E. coli AG100A and E. aerogenes (EA289 and CM64). It was followed by T. cacao and S. macrocarpon extracts which exhibited an antibacterial potential against 95% and 80% of bacterial strains, respectively. These three extracts exhibited a bactericidal effect on a few bacteria. Extracts from T. titanicus and P. dactylifera were less active as they moderately (512<MIC≤2048 µg/mL) inhibited the growth of 35% and 10% of bacteria. All extracts selectively potentiated the activities of all antibiotics with improvement activity factors (IAF) ranging from 2 to 256. G. lucida, T. cacao and S. macrocarpon potentiated the activities of 100%, 89% and 67% of antibiotics respectively against more than 70%, suggesting that they contain bioactive compounds which could be considered as efflux pumps inhibitors. Whereas T. titanicus and P. dactylifera improved the activities of almost 40% and 20% of antibiotics, respectively. This increase of activities also characterizes synergistic effects between antibiotics and these bioactive compounds. G. lucida extract at all tested concentrations, strongly inhibited the growth of bacterial strain E. coli ATCC8739 and exhibited an inhibitory effect on this bacterial H+-ATPase-mediated proton pumps increasing the pH of the medium. Conclusion: The overall results indicated that food plants among which G. lucida, T. cacao and S. macrocarpon could have a benefit interest in combatting resistant types of bacteria. Keywords: Food plants; infectious diseases; MDR bacteria; efflux pumps; antibiotics; secondary metabolites.

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Faculty Opinions recommendation of Functional vacuolar ATPase (V-ATPase) proton pumps traffic to the enterocyte brush border membrane and require CFTR.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718089195.793487140 ◽

2013 ◽

Author(s):

Richard J Naftalin

Keyword(s):

Brush Border ◽

Brush Border Membrane ◽

Vacuolar Atpase ◽

Proton Pumps

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Is the Acidic pH of the External Auditory Canal Playing a Significant Role in the Treatment of Acute Otitis Externa?

Revista de Chimie ◽

10.37358/rc.18.8.6521 ◽

2018 ◽

Vol 69 (8) ◽

pp. 2304-2305

Author(s):

Oana Ruxandra Iana ◽

Dragos Cristian Stefanescu ◽

Viorel Zainea ◽

Razvan Hainarosie

Keyword(s):

External Auditory Canal ◽

Otitis Externa ◽

Proton Pumps ◽

Acidic Ph ◽

External Ear ◽

Ph Values ◽

The World ◽

Subcutaneous Tissues ◽

Low Levels ◽

Value Changes

Variable pH values for skin have been reported in the literature, all within the acidic range, varying from 4.0 up to 7. 0. The origin of the acidic pH remains conjectural, and several factors have been incriminated with this role, such as eccrine and sebaceous secretions as well as proton pumps. Keeping low levels of pH prevents microbial dispersal as well as multiplication. The skin in the external auditory canal is also covered with this acidic mantle with antimicrobial value. Changes of pH in the external ear can lead to acute otitis externa. This condition is defined by the inflammation and infection of the cutaneous and subcutaneous tissues of the external auditory canal. 10% of the world�s population may suffer from acute otitis externa at least once in their lifetime. This paper aims to consolidate the relevance of an acidic pH in the healthy external ear and its relation to the pathogenesis and treatment of otitis externa through a prospective and interventional clinical study on 80 patients who presented to the outpatient department at Prof. Dr D. Hociota ENT Institute in Buch

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Association of radical anion with alkali cations. IV. ESR study of the 4-nitrobenzophenone radical anion

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19800369 ◽

1980 ◽

Vol 45 (2) ◽

pp. 369-375 ◽

Cited By ~ 2

Author(s):

Stanislav Miertuš ◽

Ondrej Kyseľ

Keyword(s):

Hyperfine Splitting ◽

Radical Anion ◽

Esr Spectroscopy ◽

Supporting Electrolyte ◽

Esr Spectra ◽

Proton Donor ◽

Alkali Cations

The 4-nitrobenzophenone radical anion prepared by electrolysis was studied by ESR spectroscopy. On the basis of the interpretation of ESR spectra, the conformation of this system was estimated. The effect of the concentration of supporting electrolyte and of the presence of a proton-donor agent (C2H5OH) was examined. It is assumed that changes in hyperfine splitting constants are caused by association.

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Acidification of rat liver lysosomes: quantitation and comparison with endosomes

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.4.c901 ◽

1993 ◽

Vol 265 (4) ◽

pp. C901-C917 ◽

Cited By ~ 42

Author(s):

R. W. Van Dyke

Keyword(s):

Steady State ◽

Membrane Potential ◽

Rat Liver ◽

Proton Pumps ◽

Intact Cells ◽

Early Endosomes ◽

Medium Permeability ◽

Electrogenic Proton Pump ◽

Secondary Lysosomes ◽

Permeability Studies

Both lysosomes and endosomes are acidified by an electrogenic proton pump, although studies in intact cells indicate that the steady-state internal pH (pHi) of lysosomes is more acid than that of endosomes. We undertook the present study to examine in detail the acidification mechanism of purified rat liver secondary lysosomes and to compare it with that of a population of early endosomes. Both endosomes and lysosomes exhibited ATP-dependent acidification, but proton influx rates were 2.4- to 2.7-fold greater for endosomes than for lysosomes because of differences in both buffering capacity and acidification rates, suggesting that endosomes exhibited greater numbers or rates of proton pumps. Lysosomes, however, exhibited a more acidic steady-state pHi due in part to a slower proton leak rate. Changes in medium Cl- increased acidification rates of endosomes more than lysosomes, and the lysosome ATP-dependent interior-positive membrane potential was only partially eliminated by high-Cl- medium. Permeability studies suggested that lysosomes were less permeable to Na+, Li+, and Cl- and more permeable to K+ and PO4(2-) than endosomes. Na-K-adenosine-triphosphatase did not appear to regulate acidification of either vesicle type. Endosome and lysosome acidification displayed similar inhibition profiles to N-ethylmaleimide, dicyclohexyl-carbodiimide, and vanadate, although lysosomes were somewhat more sensitive [concentration producing 50% maximal inhibition (IC50) 1 nM] to bafilomycin A1 than endosomes (IC50 7.6 nM). Oligomycin (1.5-3 microM) stimulated lysosome acidification due to shunting of membrane potential. Overall, acidification of endosomes and lysosomes was qualitatively similar but quantitatively somewhat different, possibly related to differences in the density or rate of proton pumps as well as vesicle permeability to protons, anions, and other cations.

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