Microbial rhodopsins are a class of widely studied membrane proteins that harbour a form of vitamin A as a cofactor and are therefore capable to detect visible light. These proteins have recently raised interest thanks to the application of some of them, typically the light-gated channels called channelrhodopsins, in the growing field of optogenetics. Optogenetics makes use of microbial rhodopsins to remotely activate specific neuronal cells with light, aiming to understand the function of complex neural networks in mammals and other organisms. In this thesis I propose an essay to test the function of yeast-expressed channelrhodopsins and I report on the photochemistry of the last intermediate in the photocycle of channelrhodopsin-2, with direct implications to the design of optimized optogenetic tools. Another microbial rhodopsin is bacteriorhodopsin, a light activated proton pump. As for most proteins in the rhodopsin family, proton transfers are fundamental steps in the functional mechanism of bacteriorhodopsin. Important players in proton translocation are hydrogen-bonded networks of amino acids and water molecules, and the protonation of such networks can be detected as unusually broad transient signatures in the infrared spectral range, called continuum bands. In this frame, the main contribution of this thesis work was to investigate the continuum band in bacteriorhodopsin with new approaches, based on infrared spectroscopy. We investigated the kinetics of the continuum band and compared it to the proton release and uptake from bacteriorhodopsin to and from the bulk solution. The results led us to identify two distinct hydrogenbonded networks that give rise to two continuum bands at different times during the photoreaction. The first continuum band reflects the proton release from the protein to the bulk water and further measurements with polarization-resolved spectroscopy revealed an unexpected dichroism. The protonated network is infact oriented along the membrane plane, a result that is supported by theoretical simulations. Investigations of the effect of protein solubilization in bacteriorhodopsin showed also that the membrane environment is important for the stabilization of such hydrogen-bonded networks. In conclusion these novel approaches could be applied to study continuum bands in other proteins. Chloride pumping rhodopsins are good candidates as in this thesis work I show the presence of continuum bands in two proteins of this class. Furthermore, the deep understanding of protonation dynamics via hydrogen-bonded networks is highly relevant not only for the study of microbial rhodopsins but can be applied to more complex systems.
