Структурные и функциональные исследования вирусных родопсинов тема диссертации и автореферата по ВАК РФ 03.01.02, кандидат наук Забельский Дмитрий Витальевич

• Забельский Дмитрий Витальевич
• кандидат наук
• 2022

Оглавление диссертации кандидат наук Забельский Дмитрий Витальевич

Summary

Publications

1. Literature review

1.1 Introduction

1.2 Short history of microbial rhodopsins research

1.3 Structural motifs of microbial rhodopsins

1.4 Giant viruses' diversity and importance

1.5 Viral rhodopsins

1.6 Rhodopsin-based optogenetics

1.7 Applications of optogenetics

2. Materials and Methods

2.1 Genetic constructs cloning

2.2 Expression and purification of microbial rhodopsins in Escherichia coli

2.3 Reconstitution of the protein into lipid-based systems

2.4 Crosslinking

2.5 Ion-trafficking assay with protein-containing liposomes

2.6 Time-resolved absorption spectroscopy

2.7 Crystallization

2.8 Structure determination and refinement

3 Results

3.1 Phylogenetic and functional characterization of viral rhodopsins

3.1.1 Viral rhodopsins protein selection

3.1.2 Phylogenetic analysis of viral rhodopsins (VR)

3.1.3 VR expression and purification

3.1.4 Spectroscopic characterization

3.1.5 Photocycle kinetics

3.1.6 Ion trafficking tests

3.1.7 Electrophysiological characterization

3.1.8 Optogenetical applications with VirChR1

3.1.9 Kinetic mechanism of VirChR1 calcium inhibition

3.2 Structural characterization of viral rhodopsin OLPVR1

3.2.1 Crystallization of viral rhodopsins

3.2.2 Overall structure of the OLPVR1

3.2.3 Lipid-protein interaction in OLPVR1 structure

3.2.4 Ion conducting pathway in OLPVR1

3.2.5 Conservativity analysis of viral rhodopsins

3.2.6 Retinal binding pocket and the Schiff base region of OLPVR1

3.2.7 Putative dimer form of OLPVR1

3.3 Structural characterization of OLPVRII

3.3.1 Overall structure of OLPVRII

3.3.2 Putative ion channeling properties of OLPVRII

3.3.3 Extracellular part of OLPVRII and its retinal binding pocket

4 Discussion

4.1 Structure of viral rhodopsins

4.2 Function of viral rhodopsins

4.3 Biological role of viral rhodopsins

Conclusions

Main results and the outlook

List of Abbreviations

Acknowledgments

References

Введение диссертации (часть автореферата) на тему «Структурные и функциональные исследования вирусных родопсинов»

Summary

Giant viruses are cell-size infecting agents that infect algae and encode hundreds of proteins. The unprecedented genomic complexity of giant viruses has raised questions about their role in ecological balance. Among numerous proteins encoded by giant viruses, there are two groups of viral rhodopsins, light-harvesting proteins that were never observed in other species before. In the thesis, we present a comprehensive functional and structural study of both viral rhodopsins groups. We expressed and purified protein representatives from viral rhodopsins groups and studied them using a combination of steady state and transient absorption spectroscopy, ion trafficking experiments, patch-clamp method, and X-ray crystallography.

We successfully resolved high-resolution structures of two proteins: OLPVR1 and OLPVRII from VR1 and VR2 groups respectively, that revealed unique features of viral rhodopsins, that were not previously observed in any other microbial rhodopsin structures. Functional characterization of viral rhodopsins revealed that another protein from the VR1 group, VirChR1 is a calcium-inhibited cation-conducting channelrhodopsin. We determined the kinetic mechanism of calcium inhibition that can be potentially important for fine-tuning this effect.

Besides that, we demonstrated that VirChR1 can be used for activation of neurons and therefore has potential in optogenetics, an area of neuroscience that employs light-activated protein for precise control of excitable cells. Our results suggest that viral rhodopsins are likely to be auxiliary metabolic genes that are involved in photoreception in infected host cells, and would drastically improve virus efficiency under light-accessible conditions.

Publications

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

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

3. A. Kuklin, D. Zabelskii, I. Gordeliy, J. Teixeira, A. Brûlet, V. Chupin, V. Cherezov, V. Gordeliy, On the Origin of the Anomalous Behavior of Lipid Membrane Properties in the Vicinity of the Chain-Melting Phase Transition. Sci. Rep. 10, 5749 (2020).

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

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

6. Y. L. Ryzhykau, P. S. Orekhov, M. I. Rulev, A. V. Vlasov, I. A. Melnikov, D. A. Volkov, M. Yu. Nikolaev, D. V. Zabelskii, T. N. Murugova, V. V. Chupin, A. V. Rogachev, A. Yu. Gruzinov, D. I. Svergun, M. E. Brennich, I. Yu. Gushchin, M. Soler-Lopez, A. Bothe, G. Büldt, G. Leonard, M. Engelhard, A. I. Kuklin, V. I. Gordeliy, Molecular model of a sensor of two-component signaling system. Sci. Rep. 11, 10774 (2021).

7. T. Varaksa, S. Bukhdruker, I. Grabovec, E. Marin, A. Kavaleuski, A. Gusach, K. Kovalev, I. Maslov, A. Luginina, D. Zabelskii, R. Astashkin, M. Shevtsov, S. Smolskaya, A.

Kavaleuskaya, P. Shabunya, A. Baranovsky, V. Dolgopalets, Y. Charnou, A. Savachka, R. Litvinovskaya, A. Hurski, E. Shevchenko, A. Rogachev, A. Mishin, V. Gordeliy, A. Gabrielian, D. E. Hurt, B. Nikonenko, K. Majorov, A. Apt, A. Rosenthal, A. Gilep, V. Borshchevskiy, N. Strushkevich, Metabolic Fate of Human Immunoactive Sterols in Mycobacterium tuberculosis. J. Mol. Biol. 433, 166763 (2021).

8. D. V. Zabelskii, A. V. Vlasov, Y. L. Ryzhykau, T. N. Murugova, M. Brennich, D. V. Soloviov, O. I. Ivankov, V. I. Borshchevskiy, A. V. Mishin, A. V. Rogachev, A. Round, N. A. Dencher, G. Büldt, V. I. Gordeliy, A. I. Kuklin, Ambiguities and completeness of SAS data analysis: investigations of apoferritin by SAXS/SANS EID and SEC-SAXS methods. J. Phys. Conf. Ser. 994, 012017 (2018).

Заключение диссертации по теме «Биофизика», Забельский Дмитрий Витальевич

Conclusions

In this thesis, we present a comprehensive analysis of viral rhodopsins, a distinct clade of microbial rhodopsins superfamily that consists of two groups, VR1 and VR2. Our group analyzed available metagenomic assemblies, constructed a phylogenetic tree of viral rhodopsins, and further selected multiple proteins for functional and structural characterization. Expression tests allowed us to successfully produce and optimize viral rhodopsins proteins, namely, OLPVR1, OLPVRII, and VirChR1. We tested all proteins for their expression and localization in mammalian cell lines that allowed us to electrophysiologically characterize VirChR1 rhodopsin from the VR1 group. Purified proteins were used for functional and structural characterization that allowed us to obtain photocycle kinetics of the proteins and their absorption spectra. Besides that, we tested proteins for active transport properties using pH change ion trafficking experiments. The proteins were also used crystallization in meso trials, all crystals obtained were tested for diffraction at synchrotron facilities.

All mentioned work resulted in full structural characterization of viral rhodopsins family, with structures of two proteins: OLPVR1 and OLPVRII from VR1 and VR2 groups respectively. OLPVR1 is a proteorhodopsin-like channelrhodopsin with unusual lipid molecules in the cytoplasmic part of the protein and a comprehensive hydrogen bond network inside the protein that facilitates ion transport after illumination with light. OLPVRII is a pentamer protein with a highly unusual pore assembled by highly conservative residues in the cytoplasmic part of the pentamer. This pore is highly reminiscent of a similar pore in ligand-gated ion channels that allow the assumption that proteins from the VR2 group are also ion-conducting rhodopsins.

Functional analysis of viral rhodopsins resulted in the full electrophysiological characterization of VirChR1 protein, a cation-conducting channelrhodopsin with unusual ion permeability and unique calcium inhibition. We demonstrated that VirChR1 can potentially be used for optogenetic needs as a calcium-impermeable activation tool. Further effort will be aimed to optimize the localization of viral rhodopsins in the plasma membrane of mammalian cells and to improve their closing kinetics. We used transient absorption spectra measurements to identify the kinetics mechanism of VirChR1 calcium inhibition.

Taken together, our data demonstrate that viral rhodopsins are likely to be involved in photoreception in infected host cells. Phylogeny of viral rhodopsins indicates that both

groups should have complementary functions, which is likely an ion channeling function also for OLPVRII and VR2 group.

Main results and the outlook

1) Development of an expression and purification protocol for viral rhodopsins that have reliable production of required proteins.

2) Full characterization of steady-state and transient absorption spectra of viral rhodopsins OLPVR1 and OLPVRII under different conditions.

3) Full electrophysiological characterization of VirChR1 from the VR1 group, that revealed ion channeling functions of VR1 rhodopsins. The ion selectivity tests showed that VirChR1 conducts only monovalent ions and is inhibited by divalent calcium ions.

4) VirChR1 was successfully expressed in neurons and has shown to be capable of activation of neural spike trains.

5) The transient absorbance experiments with different calcium concentrations revealed the kinetic mechanism of VirChR1 inhibition by calcium.

6) Both OLPVR1 and OLPVRII were successfully crystallized using in meso approach. The high-resolution structures of both proteins were obtained in X-ray diffraction experiments.

7) The analysis of conservativity of different amino acids among the VR family allowed us to determine key structural elements of viral rhodopsins that are involved in protein function.

Список литературы диссертационного исследования кандидат наук Забельский Дмитрий Витальевич, 2022 год

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