Bacterial proton pumps, proteorhodopsins (PRs), are a major group of light-driven membrane proteins found in marine bacteria. They are functionally and structurally distinct from archaeal and eukaryotic proton pumps. To elucidate the proton transfer mechanism by PRs and understand the differences to nonbacterial pumps on a molecular level, high-resolution structures of PRs' functional states are needed. In this work, we have determined atomic-resolution structures of MAR, a PR from marine actinobacteria, in various functional states, notably the challenging late O intermediate state. These data and information from recent atomic-resolution structures on an archaeal outward proton pump bacteriorhodopsin and bacterial inward proton pump xenorhodopsin allow for deducing key universal elements for light-driven proton pumping. First, long hydrogen-bonded chains characterize proton pathways. Second, short hydrogen bonds allow proton storage and inhibit their backflow. Last, the retinal Schiff base is the active proton donor and acceptor to and from hydrogen-bonded chains.

Belozersky Institute of Physico Chemical Biology Lomonosov Moscow State University 119991 Moscow Russia

Department of Aquatic Microbial Ecology Institute of Hydrobiology Biology Centre of the Czech Academy of Sciences 370 05 České Budějovice Czech Republic

Department of Biophysical Chemistry Max Planck Institute of Biophysics 60438 Frankfurt am Main Germany

Department of Cell and Molecular Biology Biomedical Centre Uppsala University 75124 Uppsala Sweden

Department Structural Biochemistry Max Planck Institute of Molecular Physiology 44227 Dortmund Germany

ELI Beamlines Centre ELI ERIC 252 41 Dolní Břežany Czechia

European 10 ray Free Electron Laser GmbH 22869 Schenefeld Germany

Evolutionary Genomics Group Departamento de Producción Vegetal y Microbiología Universidad Miguel Hernández San Juan de Alicante 03550 Alicante Spain

Frank Laboratory of Neutron Physics Joint Institute for Nuclear Research 141980 Dubna Russia

Hamburg Outstation c o DESY European Molecular Biology Laboratory 22607 Hamburg Germany

Institut de Biologie Structurale J P Ebel Université Grenoble Alpes CEA CNRS 38000 Grenoble France

Institute for Biophysical Chemistry Medizinische Hochschule Hannover D 30625 Hannover Germany

Institute for Nanostructure and Solid State Physics HARBOR Universität Hamburg 22761 Hamburg Germany

Laboratory of Structural Dynamics Stability and Folding of Proteins Institute of Cytology Russian Academy of Sciences 194064 St Petersburg Russia

Research Center for Molecular Mechanisms of Aging and Age Related Diseases Moscow Institute of Physics and Technology 141700 Dolgoprudny Russia

Université Paris Saclay CNRS and Ecole Normale Supérieure Paris Saclay 91190 Gif sur Yvette France

See more in PubMed

Béja O., Spudich E. N., Spudich J. L., Leclerc M., DeLong E. F., Proteorhodopsin phototrophy in the ocean. Nature 411, 786–789 (2001). PubMed

Gómez-Consarnau L., Raven J. A., Levine N. M., Cutter L. S., Wang D., Seegers B., Arístegui J., Fuhrman J. A., Gasol J. M., Sañudo-Wilhelmy S. A., Microbial rhodopsins are major contributors to the solar energy captured in the sea. Sci. Adv. 5, eaaw8855 (2019). PubMed PMC

Borshchevskiy V., Kovalev K., Round E., Efremov R., Astashkin R., Bourenkov G., Bratanov D., Balandin T., Chizhov I., Baeken C., Gushchin I., Kuzmin A., Alekseev A., Rogachev A., Willbold D., Engelhard M., Bamberg E., Büldt G., Gordeliy V., True-atomic-resolution insights into the structure and functional role of linear chains and low-barrier hydrogen bonds in proteins. Nat. Struct. Mol. Biol. 29, 440–450 (2022). PubMed

Zabelskii D., Dmitrieva N., Volkov O., Shevchenko V., Kovalev K., Balandin T., Soloviov D., Astashkin R., Zinovev E., Alekseev A., Round E., Polovinkin V., Chizhov I., Rogachev A., Okhrimenko I., Borshchevskiy V., Chupin V., Büldt G., Yutin N., Bamberg E., Koonin E., Gordeliy V., Structure-based insights into evolution of rhodopsins. Commun. Biol. 4, 821 (2021). PubMed PMC

Friedrich T., Geibel S., Kalmbach R., Chizhov I., Ataka K., Heberle J., Engelhard M., Bamberg E., Proteorhodopsin is a light-driven proton pump with variable vectoriality. J. Mol. Biol. 321, 821–838 (2002). PubMed

Zimányi L., Váró G., Chang M., Ni B., Needleman R., Lanyi J. K., Pathways of proton release in the bacteriorhodopsin photocycle. Biochemistry 31, 8535–8543 (1992). PubMed

Gushchin I., Chervakov P., Kuzmichev P., Popov A. N., Round E., Borshchevskiy V., Ishchenko A., Petrovskaya L., Chupin V., Dolgikh D. A., Arseniev A. A., Kirpichnikov M., Gordeliy V., Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria. Proc. Natl. Acad. Sci. U.S.A. 110, 12631–12636 (2013). PubMed PMC

Bergo V. B., Sineshchekov O. A., Kralj J. M., Partha R., Spudich E. N., Rothschild K. J., Spudich J. L., His-75 in proteorhodopsin, a novel component in light-driven proton translocation by primary pumps. J. Biol. Chem. 284, 2836–2843 (2009). PubMed PMC

Hempelmann F., Hölper S., Verhoefen M.-K., Woerner A. C., Köhler T., Fiedler S.-A., Pfleger N., Wachtveitl J., Glaubitz C., His75-Asp97 cluster in green proteorhodopsin. J. Am. Chem. Soc. 133, 4645–4654 (2011). PubMed

Astashkin R., Kovalev K., Bukhdruker S., Vaganova S., Kuzmin A., Alekseev A., Balandin T., Zabelskii D., Gushchin I., Royant A., Volkov D., Bourenkov G., Koonin E., Engelhard M., Bamberg E., Gordeliy V., Structural insights into light-driven anion pumping in cyanobacteria. Nat. Commun. 13, 6460 (2022). PubMed PMC

Kovalev K., Astashkin R., Gushchin I., Orekhov P., Volkov D., Zinovev E., Marin E., Rulev M., Alekseev A., Royant A., Carpentier P., Vaganova S., Zabelskii D., Baeken C., Sergeev I., Balandin T., Bourenkov G., Carpena X., Boer R., Maliar N., Borshchevskiy V., Büldt G., Bamberg E., Gordeliy V., Molecular mechanism of light-driven sodium pumping. Nat. Commun. 11, 2137 (2020). PubMed PMC

Brown L. S., Light-driven proton transfers and proton transport by microbial rhodopsins – A biophysical perspective. Biochim. Biophys. Acta Biomembr. 1864, 183867 (2022). PubMed

Efremov R., Gordeliy V. I., Heberle J., Büldt G., Time-resolved microspectroscopy on a single crystal of bacteriorhodopsin reveals lattice-induced differences in the photocycle kinetics. Biophys. J. 91, 1441–1451 (2006). PubMed PMC

Engilberge S., Caramello N., Bukhdruker S., Byrdin M., Giraud T., Jacquet P., Scortani D., Biv R., Gonzalez H., Broquet A., van der Linden P., Rose S. L., Flot D., Balandin T., Gordeliy V., Lahey-Rudolph J. M., Roessle M., de Sanctis D., Leonard G. A., Mueller-Dieckmann C., Royant A., The TR-icOS setup at the ESRF: Time-resolved microsecond UV-Vis absorption spectroscopy on protein crystals. Acta Crystallogr. D Struct. Biol. 80, 16–25 (2024). PubMed PMC

Weinert T., Skopintsev P., James D., Dworkowski F., Panepucci E., Kekilli D., Furrer A., Brünle S., Mous S., Ozerov D., Nogly P., Wang M., Standfuss J., Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography. Science 365, 61–65 (2019). PubMed

Ghai R., Mizuno C. M., Picazo A., Camacho A., Rodriguez-Valera F., Metagenomics uncovers a new group of low GC and ultra-small marine Actinobacteria. Sci. Rep. 3, 2471 (2013). PubMed PMC

Landau E. M., Rosenbusch J. P., Lipidic cubic phases: A novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci. U.S.A. 93, 14532–14535 (1996). PubMed PMC

Luecke H., Schobert B., Richter H. T., Cartailler J. P., Lanyi J. K., Structural changes in bacteriorhodopsin during ion transport at 2 angstrom resolution. Science 286, 255–260 (1999). PubMed

Luecke H., Richter H. T., Lanyi J. K., Proton transfer pathways in bacteriorhodopsin at 2.3 angstrom resolution. Science 280, 1934–1937 (1998). PubMed

Kovalev K., Tsybrov F., Alekseev A., Shevchenko V., Soloviov D., Siletsky S., Bourenkov G., Agthe M., Nikolova M., von Stetten D., Astashkin R., Bukhdruker S., Chizhov I., Royant A., Kuzmin A., Gushchin I., Rosselli R., Rodriguez-Valera F., Ilyinskiy N., Rogachev A., Borshchevskiy V., Schneider T. R., Bamberg E., Gordeliy V., Mechanisms of inward transmembrane proton translocation. Nat. Struct. Mol. Biol. 30, 970–979 (2023). PubMed

Bukhdruker S., Melnikov I., Baeken C., Balandin T., Gordeliy V., Crystallographic insights into lipid-membrane protein interactions in microbial rhodopsins. Front. Mol. Biosci. 11, 1503709 (2024). PubMed PMC

López-Pérez M., Haro-Moreno J. M., Iranzo J., Rodriguez-Valera F., Genomes of the “Candidatus Actinomarinales” order: Highly streamlined marine epipelagic actinobacteria. mSystems 5, e01041-20 (2020). PubMed PMC

Pinhassi J., DeLong E. F., Béjà O., González J. M., Pedrós-Alió C., Marine bacterial and archaeal ion-pumping rhodopsins: Genetic diversity, physiology, and ecology. Microbiol. Mol. Biol. Rev. 80, 929–954 (2016). PubMed PMC

Higuchi A., Shihoya W., Konno M., Ikuta T., Kandori H., Inoue K., Nureki O., Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping. Proc. Natl. Acad. Sci. U.S.A. 118, e2016328118 (2021). PubMed PMC

Köhler T., Weber I., Glaubitz C., Wachtveitl J., Proteorhodopsin Photocycle Kinetics Between pH 5 and pH 9. Photochem. Photobiol. 93, 762–771 (2017). PubMed

Stone K. M., Voska J., Kinnebrew M., Pavlova A., Junk M. J. N., Han S., Structural insight into proteorhodopsin oligomers. Biophys. J. 104, 472–481 (2013). PubMed PMC

Hirschi S., Kalbermatter D., Ucurum Z., Lemmin T., Fotiadis D., Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin. Nat. Commun. 12, 4107 (2021). PubMed PMC

Hirschi S., Lemmin T., Ayoub N., Kalbermatter D., Pellegata D., Ucurum Z., Gertsch J., Fotiadis D., Structural insights into the mechanism and dynamics of proteorhodopsin biogenesis and retinal scavenging. Nat. Commun. 15, 6950 (2024). PubMed PMC

Morizumi T., Ou W. L., Van Eps N., Inoue K., Kandori H., Brown L. S., Ernst O. P., X-ray crystallographic structure and oligomerization of Gloeobacter rhodopsin. Sci. Rep. 9, 11283 (2019). PubMed PMC

Iizuka A., Kajimoto K., Fujisawa T., Tsukamoto T., Aizawa T., Kamo N., Jung K.-H., Unno M., Demura M., Kikukawa T., Functional importance of the oligomer formation of the cyanobacterial H+ pump Gloeobacter rhodopsin. Sci. Rep. 9, 10711 (2019). PubMed PMC

Gordeliy V. I., Labahn J., Moukhametzianov R., Efremov R., Granzin J., Schlesinger R., Büldt G., Savopol T., Scheidig A. J., Klare J. P., Engelhard M., Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex. Nature 419, 484–487 (2002). PubMed

Gordeliy V. I., Schlesinger R., Efremov R., Büldt G., Heberle J., Crystallization in lipidic cubic phases: A case study with bacteriorhodopsin. Methods Mol. Biol. 228, 305–316 (2003). PubMed

Luecke H., Schobert B., Stagno J., Imasheva E. S., Wang J. M., Balashov S. P., Lanyi J. K., Crystallographic structure of xanthorhodopsin, the light-driven proton pump with a dual chromophore. Proc. Natl. Acad. Sci. U.S.A. 105, 16561–16565 (2008). PubMed PMC

Ran T., Ozorowski G., Gao Y., Sineshchekov O. A., Wang W., Spudich J. L., Luecke H., Cross-protomer interaction with the photoactive site in oligomeric proteorhodopsin complexes. Acta Crystallogr. D Biol. Crystallogr. 69, 1965–1980 (2013). PubMed

Chazan A., Das I., Fujiwara T., Murakoshi S., Rozenberg A., Molina-Márquez A., Sano F. K., Tanaka T., Gómez-Villegas P., Larom S., Pushkarev A., Malakar P., Hasegawa M., Tsukamoto Y., Ishizuka T., Konno M., Nagata T., Mizuno Y., Katayama K., Abe-Yoshizumi R., Ruhman S., Inoue K., Kandori H., León R., Shihoya W., Yoshizawa S., Sheves M., Nureki O., Béjà O., Phototrophy by antenna-containing rhodopsin pumps in aquatic environments. Nature 615, 535–540 (2023). PubMed

Reckel S., Gottstein D., Stehle J., Löhr F., Verhoefen M.-K., Takeda M., Silvers R., Kainosho M., Glaubitz C., Wachtveitl J., Bernhard F., Schwalbe H., Güntert P., Dötsch V., Solution NMR Structure of Proteorhodopsin. Angew. Chem. Int. Ed. Engl. 50, 11942–11946 (2011). PubMed PMC

Gao K., Beardall J., Häder D.-P., Hall-Spencer J. M., Gao G., Hutchins D. A., Effects of ocean acidification on marine photosynthetic organisms under the concurrent influences of warming, UV radiation, and deoxygenation. Front. Mar. Sci. 6, 322 (2019).

Imasheva E. S., Balashov S. P., Wang J. M., Lanyi J. K., pH-dependent transitions in xanthorhodopsin. Photochem. Photobiol. 82, 1406–1413 (2006). PubMed PMC

Balashov S. P., Petrovskaya L. E., Lukashev E. P., Imasheva E. S., Dioumaev A. K., Wang J. M., Sychev S. V., Dolgikh D. A., Rubin A. B., Kirpichnikov M. P., Lanyi J. K., Aspartate-histidine interaction in the retinal schiff base counterion of the light-driven proton pump of Exiguobacterium sibiricum. Biochemistry 51, 5748–5762 (2012). PubMed PMC

Okumura H., Murakami M., Kouyama T., Crystal structures of acid blue and alkaline purple forms of bacteriorhodopsin. J. Mol. Biol. 351, 481–495 (2005). PubMed

Dioumaev A. K., Brown L. S., Shih J., Spudich E. N., Spudich J. L., Lanyi J. K., Proton transfers in the photochemical reaction cycle of proteorhodopsin. Biochemistry 41, 5348–5358 (2002). PubMed

Subramanlam S., Henderson R., Molecular mechanism of vectorial proton translocation by bacteriorhodopsin. Nature 406, 653–657 (2000). PubMed

Huber R., Köhler T., Lenz M. O., Bamberg E., Kalmbach R., Engelhard M., Wachtveitl J., pH-dependent photoisomerization of retinal in proteorhodopsin. Biochemistry 44, 1800–1806 (2005). PubMed

Lenz M. O., Huber R., Schmidt B., Gilch P., Kalmbach R., Engelhard M., Wachtveitl J., First steps of retinal photoisomerization in proteorhodopsin. Biophys. J. 91, 255–262 (2006). PubMed PMC

Ikeda D., Furutani Y., Kandori H., FTIR study of the retinal Schiff base and internal water molecules of proteorhodopsin. Biochemistry 46, 5365–5373 (2007). PubMed

Mehler M., Eckert C. E., Leeder A. J., Kaur J., Fischer T., Kubatova N., Brown L. J., Brown R. C. D., Becker-Baldus J., Wachtveitl J., Glaubitz C., Chromophore distortions in photointermediates of proteorhodopsin visualized by dynamic nuclear polarization-enhanced solid-state NMR. J. Am. Chem. Soc. 139, 16143–16153 (2017). PubMed

Bada Juarez J. F., Judge P. J., Adam S., Axford D., Vinals J., Birch J., Kwan T. O. C., Hoi K. K., Yen H.-Y., Vial A., Milhiet P.-E., Robinson C. V., Schapiro I., Moraes I., Watts A., Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization. Nat. Commun. 12, 629 (2021). PubMed PMC

Zabelskii D., Alekseev A., Kovalev K., Rankovic V., Balandin T., Soloviov D., Bratanov D., Savelyeva E., Podolyak E., Volkov D., Vaganova S., Astashkin R., Chizhov I., Yutin N., Rulev M., Popov A., Eria-Oliveira A.-S., Rokitskaya T., Mager T., Antonenko Y., Rosselli R., Armeev G., Shaitan K., Vivaudou M., Büldt G., Rogachev A., Rodriguez-Valera F., Kirpichnikov M., Moser T., Offenhäusser A., Willbold D., Koonin E., Bamberg E., Gordeliy V., Viral rhodopsins 1 are an unique family of light-gated cation channels. Nat. Commun. 11, 5707 (2020). PubMed PMC

Park J. H., Scheerer P., Hofmann K. P., Choe H.-W., Ernst O. P., Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008). PubMed

Hilf R. J. C., Dutzler R., Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel. Nature 457, 115–118 (2009). PubMed

Fujisawa T., Nishikawa K., Tamogami J., Unno M., Conformational analysis of a retinal schiff base chromophore in proteorhodopsin by Raman optical activity. J. Phys. Chem. Lett. 12, 9564–9568 (2021). PubMed

Bamann C., Bamberg E., Wachtveitl J., Glaubitz C., Proteorhodopsin. Biochim. Biophys. Acta Bioenerg. 1837, 614–625 (2014). PubMed

Schätzler B., Dencher N. A., Tittor J., Oesterhelt D., Yaniv-Checover S., Nachliel E., Gutman M., Subsecond proton-hole propagation in bacteriorhodopsin. Biophys. J. 84, 671–686 (2003). PubMed PMC

Faramarzi S., Feng J., Mertz B., Allosteric effects of the proton donor on the microbial proton pump Proteorhodopsin. Biophys. J. 115, 1240–1250 (2018). PubMed PMC

Sasaki S., Tamogami J., Nishiya K., Demura M., Kikukawa T., Replaceability of Schiff base proton donors in light-driven proton pump rhodopsins. J. Biol. Chem. 297, 101013 (2021). PubMed PMC

Nagle J. F., Morowitz H. J., Molecular mechanisms for proton transport in membranes. Proc. Natl. Acad. Sci. U.S.A. 75, 298–302 (1978). PubMed PMC

Nagle J. F., Tristram-Nagle S., Hydrogen bonded chain mechanisms for proton conduction and proton pumping. J. Membr. Biol. 74, 1–14 (1983). PubMed

Noji T., Chiba Y., Saito K., Ishikita H., Energetics of the H-bond network in Exiguobacterium sibiricum rhodopsin. Biochemistry 63, 1505–1512 (2024). PubMed PMC

Freier E., Wolf S., Gerwert K., Proton transfer via a transient linear water-molecule chain in a membrane protein. Proc. Natl. Acad. Sci. U.S.A. 108, 11435–11439 (2011). PubMed PMC

Miranda M. R. M., Choi A. R., Shi L., Bezerra A. G. Jr., Jung K.-H., Brown L. S., The photocycle and proton translocation pathway in a cyanobacterial ion-pumping rhodopsin. Biophys. J. 96, 1471–1481 (2009). PubMed PMC

Garczarek F., Gerwert K., Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy. Nature 439, 109–112 (2006). PubMed

Sugimoto T., Katayama K., Kandori H., FTIR study of light-induced proton transfer and Ca2+ binding in T82D mutant of TAT rhodopsin. Biophys. J. 123, 4245–4255 (2024). PubMed PMC

Goyal P., Ghosh N., Phatak P., Clemens M., Gaus M., Elstner M., Cui Q., Proton storage site in bacteriorhodopsin: New insights from quantum mechanics/molecular mechanics simulations of microscopic pKa and infrared spectra. J. Am. Chem. Soc. 133, 14981–14997 (2011). PubMed PMC

Maag D., Mast T., Elstner M., Cui Q., Kubař T., O to bR transition in bacteriorhodopsin occurs through a proton hole mechanism. Proc. Natl. Acad. Sci. U.S.A. 118, e2024803118 (2021). PubMed PMC

Shigeta A., Ito S., Inoue K., Okitsu T., Wada A., Kandori H., Kawamura I., Solid-state nuclear magnetic resonance structural study of the retinal-binding pocket in sodium ion pump rhodopsin. Biochemistry 56, 543–550 (2017). PubMed

Caramello N., Royant A., From femtoseconds to minutes: Time-resolved macromolecular crystallography at XFELs and synchrotrons. Acta Crystallogr. D Struct. Biol. 80, 60–79 (2024). PubMed PMC

Khusainov G., Standfuss J., Weinert T., The time revolution in macromolecular crystallography. Struct. Dyn. 11, 020901 (2024). PubMed PMC

Gushchin I., Reshetnyak A., Borshchevskiy V., Ishchenko A., Round E., Grudinin S., Engelhard M., Büldt G., Gordeliy V., Active state of sensory rhodopsin II: Structural determinants for signal transfer and proton pumping. J. Mol. Biol. 412, 591–600 (2011). PubMed

Liu S., Li W., Protein fusion strategies for membrane protein stabilization and crystal structure determination. Crystals 12, 1041 (2022).

Dai S., Funk L.-M., von Pappenheim F. R., Sautner V., Paulikat M., Schröder B., Uranga J., Mata R. A., Tittmann K., Low-barrier hydrogen bonds in enzyme cooperativity. Nature 573, 609–613 (2019). PubMed

Ogata H., Nishikawa K., Lubitz W., Hydrogens detected by subatomic resolution protein crystallography in a [NiFe] hydrogenase. Nature 520, 571–574 (2015). PubMed

Woińska M., Grabowsky S., Dominiak P. M., Woźniak K., Jayatilaka D., Hydrogen atoms can be located accurately and precisely by X-ray crystallography. Sci. Adv. 2, e1600192 (2016). PubMed PMC

Eriksson U. K., Fischer G., Friemann R., Enkavi G., Tajkhorshid E., Neutze R., Subangstrom resolution X-ray structure details aquaporin-water interactions. Science 340, 1346–1349 (2013). PubMed PMC

Blakeley M. P., Hasnain S. S., Antonyuk S. V., Sub-atomic resolution X-ray crystallography and neutron crystallography: Promise, challenges and potential. IUCrJ 2, 464–474 (2015). PubMed PMC

Fuhrman J. A., Schwalbach M. S., Stingl U., Proteorhodopsins: An array of physiological roles? Nat. Rev. Microbiol. 6, 488–494 (2008). PubMed

Neutze R., Pebay-Peyroula E., Edman K., Royant A., Navarro J., Landau E. M., Bacteriorhodopsin: A high-resolution structural view of vectorial proton transport. Biochim. Biophys. Acta 1565, 144–167 (2002). PubMed

Inoue K., Ito S., Kato Y., Nomura Y., Shibata M., Uchihashi T., Tsunoda S. P., Kandori H., A natural light-driven inward proton pump. Nat. Commun. 7, 13415 (2016). PubMed PMC

Shevchenko V., Mager T., Kovalev K., Polovinkin V., Alekseev A., Juettner J., Chizhov I., Bamann C., Vavourakis C., Ghai R., Gushchin I., Borshchevskiy V., Rogachev A., Melnikov I., Popov A., Balandin T., Rodriguez-Valera F., Manstein D. J., Bueldt G., Bamberg E., Gordeliy V., Inward H+ pump xenorhodopsin: Mechanism and alternative optogenetic approach. Sci. Adv. 3, e1603187 (2017). PubMed PMC

Okuyama A., Hososhima S., Kandori H., Tsunoda S. P., Driving forces of proton-pumping rhodopsins. Biophys. J. 123, 4274–4284 (2024). PubMed PMC

Warshel A., Papazyan A., Kollman P. A., On low-barrier hydrogen bonds and enzyme catalysis. Science 269, 102–106 (1995). PubMed

Yamaguchi S., Kamikubo H., Kurihara K., Kuroki R., Niimura N., Shimizu N., Yamazaki Y., Kataoka M., Low-barrier hydrogen bond in photoactive yellow protein. Proc. Natl. Acad. Sci. U.S.A. 106, 440–444 (2009). PubMed PMC

Wang J., Visualization of H atoms in the X-ray crystal structure of photoactive yellow protein: Does it contain low-barrier hydrogen bonds? Protein Sci. 28, 1966–1972 (2019). PubMed PMC

Katoh K., Standley D. M., MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013). PubMed PMC

Minh B. Q., Schmidt H. A., Chernomor O., Schrempf D., Woodhams M. D., Von Haeseler A., Lanfear R., Teeling E., IQ-TREE 2: New models and efficient methods for phylogenetic inference in the Genomic Era. Mol. Biol. Evol. 37, 1530–1534 (2020). PubMed PMC

Kalyaanamoorthy S., Minh B. Q., Wong T. K. F., Von Haeseler A., Jermiin L. S., ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587–589 (2017). PubMed PMC

Hoang D. T., Chernomor O., Von Haeseler A., Minh B. Q., Vinh L. S., UFBoot2: Improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2018). PubMed PMC

Edgar R. C., MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004). PubMed PMC

Okonechnikov K., Golosova O., Fursov M., UGENE team , Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics 28, 1166–1167 (2012). PubMed

Letunic I., Bork P., Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293–W296 (2021). PubMed PMC

Studier F. W., Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005). PubMed

Renault L., Guibert B., Cherfils J., Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor. Nature 426, 525–530 (2003). PubMed

Gushchin I., Shevchenko V., Polovinkin V., Kovalev K., Alekseev A., Round E., Borshchevskiy V., Balandin T., Popov A., Gensch T., Fahlke C., Bamann C., Willbold D., Büldt G., Bamberg E., Gordeliy V., Crystal structure of a light-driven sodium pump. Nat. Struct. Mol. Biol. 22, 390–395 (2015). PubMed

Bamberg E., Apell H.-J., Dencher N. A., Sperling W., Stieve H., Läuger P., Photocurrents generated by bacteriorhodopsin on planar bilayer membranes. Biophys. Struct. Mech. 5, 277–292 (1979).

Chizhov I., Chernavskii D. S., Engelhard M., Mueller K. H., Zubov B. V., Hess B., Spectrally silent transitions in the bacteriorhodopsin photocycle. Biophys. J. 71, 2329–2345 (1996). PubMed PMC

Chizhov I., Schmies G., Seidel R., Sydor J. R., Lüttenberg B., Engelhard M., The photophobic receptor from natronobacterium pharaonis: Temperature and pH dependencies of the photocycle of sensory Rhodopsin II. Biophys. J. 75, 999–1009 (1998). PubMed PMC

Chizhov I., Engelhard M., Temperature and halide dependence of the photocycle of halorhodopsin from Natronobacterium pharaonis. Biophys. J. 81, 1600–1612 (2001). PubMed PMC

Bratanov D., Kovalev K., Machtens J.-P., Astashkin R., Chizhov I., Soloviov D., Volkov D., Polovinkin V., Zabelskii D., Mager T., Gushchin I., Rokitskaya T., Antonenko Y., Alekseev A., Shevchenko V., Yutin N., Rosselli R., Baeken C., Borshchevskiy V., Bourenkov G., Popov A., Balandin T., Büldt G., Manstein D. J., Rodriguez-Valera F., Fahlke C., Bamberg E., Koonin E., Gordeliy V., Unique structure and function of viral rhodopsins. Nat. Commun. 10, 4939 (2019). PubMed PMC

Volkov O., Kovalev K., Polovinkin V., Borshchevskiy V., Bamann C., Astashkin R., Marin E., Popov A., Balandin T., Willbold D., Büldt G., Bamberg E., Gordeliy V., Structural insights into ion conduction by channelrhodopsin 2. Science 358, eaan8862 (2017). PubMed

Kovalev K., Volkov D., Astashkin R., Alekseev A., Gushchin I., Haro-Moreno J. M., Chizhov I., Siletsky S., Mamedov M., Rogachev A., Balandin T., Borshchevskiy V., Popov A., Bourenkov G., Bamberg E., Rodriguez-Valera F., Büldt G., Gordeliy V., High-resolution structural insights into the heliorhodopsin family. Proc. Natl. Acad. Sci. U.S.A. 117, 4131–4141 (2020). PubMed PMC

Kabsch W., XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010). PubMed PMC

Günther S., Reinke P. Y. A., Fernández-García Y., Lieske J., Lane T. J., Ginn H. M., Koua F. H. M., Ehrt C., Ewert W., Oberthuer D., Yefanov O., Meier S., Lorenzen K., Krichel B., Kopicki J.-D., Gelisio L., Brehm W., Dunkel I., Seychell B., Gieseler H., Norton-Baker B., Escudero-Pérez B., Domaracky M., Saouane S., Tolstikova A., White T. A., Hänle A., Groessler M., Fleckenstein H., Trost F., Galchenkova M., Gevorkov Y., Li C., Awel S., Peck A., Barthelmess M., Schlünzen F., Xavier P. L., Werner N., Andaleeb H., Ullah N., Falke S., Srinivasan V., França B. A., Schwinzer M., Brognaro H., Rogers C., Melo D., Zaitseva-Kinneberg J. I., Knoska J., Peña-Murillo G. E., Mashhour A. R., Hennicke V., Fischer P., Hakanpää J., Meyer J., Gribbon P., Ellinger B., Kuzikov M., Wolf M., Beccari A. R., Bourenkov G., von Stetten D., Pompidor G., Bento I., Panneerselvam S., Karpics I., Schneider T. R., Garcia-Alai M. M., Niebling S., Günther C., Schmidt C., Schubert R., Han H., Boger J., Monteiro D. C. F., Zhang L., Sun X., Pletzer-Zelgert J., Wollenhaupt J., Feiler C. G., Weiss M. S., Schulz E.-C., Mehrabi P., Karničar K., Usenik A., Loboda J., Tidow H., Chari A., Hilgenfeld R., Uetrecht C., Cox R., Zaliani A., Beck T., Rarey M., Günther S., Turk D., Hinrichs W., Chapman H. N., Pearson A. R., Betzel C., Meents A., X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease. Science 372, 642–646 (2021). PubMed PMC

Słabicki M., Kozicka Z., Petzold G., Li Y.-D., Manojkumar M., Bunker R. D., Donovan K. A., Sievers Q. L., Koeppel J., Suchyta D., Sperling A. S., Fink E. C., Gasser J. A., Wang L. R., Corsello S. M., Sellar R. S., Jan M., Gillingham D., Scholl C., Fröhling S., Golub T. R., Fischer E. S., Thomä N. H., Ebert B. L., The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K. Nature 585, 293–297 (2020). PubMed PMC

Andreeva L., Hiller B., Kostrewa D., Lässig C., De Oliveira Mann C. C., Jan Drexler D., Maiser A., Gaidt M., Leonhardt H., Hornung V., Hopfner K.-P., cGAS senses long and HMGB/TFAM-bound U-turn DNA by forming protein–DNA ladders. Nature 549, 394–398 (2017). PubMed

Sievers Q. L., Petzold G., Bunker R. D., Renneville A., Słabicki M., Liddicoat B. J., Abdulrahman W., Mikkelsen T., Ebert B. L., Thomä N. H., Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 362, eaat0572 (2018). PubMed PMC

Evans P., Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006). PubMed

Vagin A., Teplyakov A., Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010). PubMed

Melnikov I., Polovinkin V., Kovalev K., Gushchin I., Shevtsov M., Shevchenko V., Mishin A., Alekseev A., Rodriguez-Valera F., Borshchevskiy V., Cherezov V., Leonard G. A., Gordeliy V., Popov A., Fast iodide-SAD phasing for high-throughput membrane protein structure determination. Sci. Adv. 3, e1602952 (2017). PubMed PMC

Amor J. C., Harrison D. H., Kahn R. A., Ringe D., Structure of the human ADP-ribosylation factor 1 complexed with GDP. Nature 372, 704–708 (1994). PubMed

Terwilliger T. C., Grosse-Kunstleve R. W., Afonine P. V., Moriarty N. W., Zwart P. H., Hung L. W., Read R. J., Adams P. D., Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69 (2007). PubMed PMC

Emsley P., Lohkamp B., Scott W. G., Cowtan K., Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010). PubMed PMC

Murshudov G. N., Skubák P., Lebedev A. A., Pannu N. S., Steiner R. A., Nicholls R. A., Winn M. D., Long F., Vagin A. A., REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011). PubMed PMC

Afonine P. V., Grosse-Kunstleve R. W., Echols N., Headd J. J., Moriarty N. W., Mustyakimov M., Terwilliger T. C., Urzhumtsev A., Zwart P. H., Adams P. D., Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012). PubMed PMC

Williams C. J., Headd J. J., Moriarty N. W., Prisant M. G., Videau L. L., Deis L. N., Verma V., Keedy D. A., Hintze B. J., Chen V. B., Jain S., Lewis S. M., Arendall W. B. III, Snoeyink J., Adams P. D., Lovell S. C., Richardson J. S., Richardson D. C., MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018). PubMed PMC

Diederichs K., Karplus P. A., Better models by discarding data? Acta Crystallogr. D Biol. Crystallogr. 69, 1215–1222 (2013). PubMed PMC

Ho B. K., Gruswitz F., HOLLOW: Generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8, 49 (2008). PubMed PMC

Lomize M. A., Pogozheva I. D., Joo H., Mosberg H. I., Lomize A. L., OPM database and PPM web server: Resources for positioning of proteins in membranes. Nucleic Acids Res. 40, D370–D376 (2012). PubMed PMC

De Zitter E., Coquelle N., Oeser P., Barends T. R. M., Colletier J.-P., Xtrapol8 enables automatic elucidation of low-occupancy intermediate-states in crystallographic studies. Commun. Biol. 5, 640 (2022). PubMed PMC

Cruickshank D. W., Remarks about protein structure precision. Acta Crystallogr. D Biol. Crystallogr. 55, 583–601 (1999). PubMed

G. A. J. Jeffrey, G. A. Jeffrey, An Introduction to Hydrogen Bonding (Oxford Univ. Press, 1997).

P. A. Frey, Encyclopedia of Biological Chemistry (Elsevier, 2004).

von Stetten D., Giraud T., Carpentier P., Sever F., Terrien M., Dobias F., Juers D. H., Flot D., Mueller-Dieckmann C., Leonard G. A., de Sanctis D., Royant A., In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF. Acta Crystallogr. D Biol. Crystallogr. 71, 15–26 (2015). PubMed PMC

Smith S. O., Lugtenburg J., Mathies R. A., Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy. J. Membr. Biol. 85, 95–109 (1985). PubMed

Kouyama T., Ihara K., Existence of two substates in the O intermediate of the bacteriorhodopsin photocycle. Biochim. Biophys. Acta Biomembr. 1864, 183998 (2022). PubMed

Heberle J., Proton transfer reactions across bacteriorhodopsin and along the membrane. Biochim. Biophys. Acta 1458, 135–147 (2000). PubMed

Kandori H., Hydration switch model for the proton transfer in the Schiff base region of bacteriorhodopsin. Biochim. Biophys. Acta 1658, 72–79 (2004). PubMed

Cao Z., Peng Y., Yan T., Li S., Li A., Voth G. A., Mechanism of fast proton transport along one-dimensional water chains confined in carbon nanotubes. J. Am. Chem. Soc. 132, 11395–11397 (2010). PubMed

Deamer D. W., Proton permeation of lipid bilayers. J. Bioenerg. Biomembr. 19, 457–479 (1987). PubMed