New chloride and proton light-driven pumps : functional characterization and crystallization

The proteins can be divided into the two major types in terms of the cell localization: membrane proteins and non-membrane proteins. Membrane proteins typically constitute about third of all proteins that cell expresses in a given moment and play important role in a cell's life. So, the membrane protein receptors mediate information flow from the environment into the cell modulating the cell mechanisms in response to the environmental changes. Another type of membrane proteins - the transport proteins - allow cells to communicate by means of signaling molecules and receive necessary nutrients from the environment. The vast studies led to a new method being called the optogenetics. This new approach utilizes the light to control cells in a living tissue. The key elements of this method are the membrane light sensitive proteins that work as the "function switches". And the majority of such "switches" belongs to the rhodopsins. Unlike direct neuron excitation methods via electrodes, the optogenetic approach is less invasive. The rhodopsin gene is injected into the cell allowing latter to express the rhodopsin and to embed it into the membrane the same way as its endogenous proteins. After the injection cell becomes light-sensitive and can be manipulated just with the visible light impulses. The rhodopsins are known for a while and estimated to consist of two major types: microbial rhodopsins (type I) and mammalian rhodopsins (type II). Despite the common name (rhodopsin, derived from Greek for "rose" and "sight"), mammalian rhodopsins significantly differ from those in microorganisms - they belong to a large class of proteins called GPCRs, while their microbial analogues don't. Due to the differences in mechanisms of function, microbial rhodopsins are simpler thus are more convenient to use in the neuroscience. The optogenetics uses rhodopsins to investigate the cells' mechanisms and their interaction within the tissue. But each protein has its own characteristics and there's no ideal "instrument" for all the applications - each experiment requires the specific "tools" (rhodopsins) with different properties for optogenetic investigations. So, one experiment requires fast quenching of neuronal cells signaling thus having no particular requirements to a long-term behavior of the protein, while in the other experiment investigator may use rhodopsins with slow kinetics, but requires the moderately high steady-state signal during the long-term illumination. The large diversity of possible optogenetic applications requires the expansion of a "tool-kit", which in turn leads to a characterization of the new rhodopsins.The most abundantly used rhodopsins in optogenetics are channels (ChRs, derived from channel rhodopsins). These channels provide a light-modulated passive transport of ions along the gradient, which leads to a neuronal membrane depolarization. The other proteins type being applied is microbial pumping rhodopsins (the sensory rhodopsins despite being reported to be successfully applied need further characterization). The microbial pumping rhodopsins can be divided into two major groups: cation pumps and anion pumps. The first includes sodium pumps and proton pumps. The optogenetic application of latter is still disputed due to their ability to directly change the pH, while first posses low selectivity/overall poor performance. Thus, halorhodopsins (anion pumping rhodopsins, HRs), despite the debates on their ability to hyperpolarize membrane beyond the physiological levels, are more common membrane repolarizating rhodopsins. The main goal of this work was to expand the list of rhodopsins that might be used as the membrane repolarization tools in terms of optogenetic. The genes of non-characterised before rhodopsins were optimised and the optimal conditions for rhodopsins expression in E.coli expression system were estimated. The positive results of expression trials of these membrane proteins were as well demonstrated for relatively new LEXSY expression system. The two new rhodopsins PGr1 and PMr2 were expressed in E.coli and characterised from kinetic point of view. The model of unidirectional transitions cycle was proposed. The functional activity of a novel anion pump with significantly shifted into the blue light region (in compare to the reported halorhodopsins) was demonstrated as well as the pumping activity of a new proton-pumping rhodopsin was proven. Along with functional characterisation, the importance of few structural features of novel anion pump was shown. The crystals of proton pump were obtained and the crystallisation trials for both the proton pump and anion pump are still going