Dissertation, RWTH Aachen University, 2020; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2021). = Dissertation, RWTH Aachen University, 2020, Light-driven proton and chloride pumps were discovered as proteins used by halophilic archaea to produce energy phototrophically. A few decades later light-driven cation channel was described. Its functional characteristics were appropriate to be able to induce depolarizing currents in neural cells, that lead to their activation upon illumination. That was the start of optogenetics. 5 years later, in 2010 optogenetics will be named “the method of the year”. And now, 10 years from that moment, optogenetics became a rapidly developing and expanding tool in neuroscience and beyond that. Started with a toolkit of three functionally distinct proteins (family of bacteriorhodopsin-like proton pumps, chloride pump halorhodopsin and cation transporting channelrhodopsin 2), optogenetics requires new, optimized and tuned tools. That lead to rational mutagenesis of known proteins to affect the pumping rate, spectral absorption optimum, recovery after continuous illumination. The use of available tools left some concerns, like proton pumping might change the pH value around the cells illuminated and therefore unnecessarily activate regulation proteins; or tighter control may be desired on the number of ions transported, not like just to open a pore. New genome sequencing technologies led to metagenomics, the analysis of genetic content of a sample from wild environment. It appeared that rhodopsins, light-driven ions transporters/sensors, are widespread among all taxa. Around 30 different major groups (clades) are identified by now. Representatives from some clades are being picked up as candidates that can be used in optogenetic applications. They are now termed as “next generation optogenetic tools”. The present work is about three clades of rhodopsins that are promising candidates for the expanded optogenetic toolkit. This thesis accumulates the data from year 2015. The first discovered representative of light-driven sodium pumping rhodopsins Dokdonia eikasta KR2 is characterized functionally. After the solution of crystal structure* rational mutagenesis was successfully done in order to engineer a protein that is pumping potassium ions upon illumination. Characterization of the mutant included functional pumping tests in Escherichia coli suspensions and electrophysiological measurements on single bilayer black lipid membranes.The importance of the study of sodium pumping rhodopsin and engineered potassium pumping is high. These ions are natural in the process of nerve cells activation, i.e. exactly these ions are used by neurons to fire. Second rhodopsin in the present work is a very small proton pump, proteorhodopsin homologue, from an ultra-small actinobacterium Candidatus actinomarina minuta, rhodopsin MacR. Optogenetic applications with MacR were of limited success, however the protein demonstrated extreme crystallizability giving high order diffraction pattern (the crystal structure* was solved at 1.4 Å). Finally, a member of xenorhodopsin clade from Nanosalina XeR was extensively characterized. Although being a homologue to a sensory rhodopsin, xenorhodopsins appeared to be inward proton pumping proteins. NsXeR was comprehensively characterized, starting the target gene identification, through functional and spectroscopic characterization, followed by electrophysiological measurements in HEK293 and NG108 cells. XeRs ability to activate neurons was also demonstrated at a high frequency (50 Hz). NsXeR’s crystal structure* is also described in the present work. Primary results of the present thesis are published in high-ranked journals: Crystal structure of light-driven sodium pump in Nature Structural and Molecular Biology, 2015Inward H+ pump xenorhodopsin: Mechanism and alternative optogenetic approach in Science Advances, 2017Fast iodide-SAD phasing for high-throughput membrane protein structure determination in Science Advances, 2017To sum up, the present work led to the addition of new promising rhodopsins to the optogenetic toolkit., Published by RWTH Aachen University, Aachen