New Light-Driven Channels and Transporters for Optogenetics
Final Report Abstract
Optogenetics – enabling the control of cell function by light through the use of light-activated proteins – is a novel multidisciplinary science and biotechnology field that integrates electrophysiology, structural biology, optics, electronics and genetic engineering. Optogenetics promises to solve a challenging problem of cellular control by enabling unprecedented temporal and spatial precision in tissues and animals, which electrical stimulations and pharmacological methods are not able to achieve. It offers a breakthrough in our ability to study the mechanisms of complex processes in neuronal circuits, such as learning and motor function. It also brings a promise of providing an entirely new approach to the restoration of function in blindness or brain degeneration, and to the treatment of a variety of other neurological and mental disorders. The major objective of the project is the discovery and design of light-driven proteins with novel properties, suitable for optogenetic studies of biological systems. This implies the screening of genomic data, expression of novel membrane proteins in various tissues and the functional characterization and structural analysis. Bioinformatics techniques were used to extract sequences of opsin-like proteins from the published genomes of new microbial species. For structural studies, these proteins were overexpressed in suitable cells, purified in large quantity, and crystallized to resolve their atomic structures using synchrotron sources. To assess the function of the selected proteins, they were expressed in model cells and characterized with electrophysiological methods. Optogenetic tests were conducted in native cells expressing optimized versions of the proteins. Project main results: Structure of ChannelRhodopsin 2 ChR2: We reported in Science the high-resolution structures of ChR2 wild type in its dark-adapted state and of a ChR mutant. The structures provide a detailed understanding of site-specific mutations used in optogenetics and will enable the rational design of optimized optogenetic tools. Viral rhodopsins: Rhodopsins are also encoded by viruses. We cloned, expressed functionally and solved the structure of several viral rhodopsins. These studies uncovered original properties of fundamental and practical interest in optogenetics. Rhodopsins were used in animal models to characterize neuronal function.
Publications
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(2016) Structure of the light-driven sodium pump KR2 and its implications for optogenetics. FEBS J. 283:1232-8
Gushchin I, Shevchenko V, Polovinkin V, Borshchevskiy V, Buslaev P, Bamberg E, Gordeliy V
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(2017) Inward H+ pump xenorhodopsin: Mechanism and alternative optogenetic approach. Sci Adv. 3:e1603187
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 DJ, Bueldt G, Bamberg E, Gordeliy V
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(2017) Structural insights into ion conduction by channelrhodopsin 2. Science. 358:pii =eaan8862
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
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(2018) Aspergillus fumigatus Infection-Induced Neutrophil Recruitment and Location in the Conducting Airway of Immunocompetent, Neutropenic, and Immunosuppressed Mice. J Immunol Res. 2018:5379085
Shevchenko MA, Bogorodskiy AO, Troyanova NI, Servuli EA, Bolkhovitina EL, Büldt G, Fahlke C, Gordeliy VI, Gensch T, Borshchevskiy VI, Sapozhnikov AM
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(2018) Efficient non-cytotoxic fluorescent staining of halophiles. Sci Rep. 8:2549
Maslov I, Bogorodskiy A, Mishin A, Okhrimenko I, Gushchin I, Kalenov S, Dencher NA, Fahlke C, Büldt G, Gordeliy V, Gensch T, Borshchevskiy V
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(2019) Structure and mechanisms of sodium-pumping KR2 rhodopsin. Sci Adv. 5:eaav2671
Kovalev K, Polovinkin V, Gushchin I, Alekseev A, Shevchenko V, Borshchevskiy V, Astashkin R, Balandin T, Bratanov D, Vaganova S, Popov A, Chupin V, Büldt G, Bamberg E, Gordeliy V
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(2019) Unique structure and function of viral rhodopsins. Nat Commun. 10:4939
Bratanov D, Kovalev K, Machtens JP, 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 DJ, Rodriguez-Valera F, Fahlke C, Bamberg E, Koonin E, Gordeliy V
