Channelrhodopsin (ChR) is a fundamental transmebrane protein (TMP) that can act as light-sensitive ion channel through the cell membrane. This protein performs its function through a series of conformational changes triggered by the absorption of visible light. In the last decade, ChR has been widely studied for its possible applications in the field of optogenetics but the functional mechanisms related to its function is still partly unknown.
Mid-Infrared (IR) spectroscopy is considered a powerful label-free tool to investigate the modifications of light-sensitive TMPs structure. However, the sensitivity of mid-IR spectroscopy, in terms of probed molecules, requires to be applied to large amount of purified proteins in order to probe the subtle light-induced modifications of protein IR absorption spectrum. In order to study the more biologically interesting case of proteins embedded in native cell membranes, which are intrinsically heterogeneous, it is necessary to push IR spectroscopy capabilities at the nanoscale exploiting plasmonic approaches, such as in the novel IR platform based on an Atomic Force Microscopy (AFM) coupled to a quantum cascade laser. This technique allows one to combine spectroscopic information with nanometric resolution provided by the scanning probe approach (AFM).
Despite IR nanospectroscopy has been largely applied to protein samples, the first application of this approach to the investigation of the light-sensitive TMPs has been demonstrated only recently with experiments carried out during my Master degree and first year of PhD project. I have investigated the light-induced activity of Bacteriorhodopsin, a prototype for light-sensitive TMPs, at the single cell membrane monolayer. These results pave the way towards the investigation at the nanoscale of more interesting light-sensitive optogenetic gates, such as ChR, in order to better understand the functional mechanisms of these proteins when embedded in their native cell membrane.
The innovation of my research project is to bring at the nanoscale the spectroscopic study of the light-sensitive TMP Channelrhodopsin (ChR). So far, IR difference spectroscopy has been one of the most important source of information on protein structural changes. In particular the blue light starts the ChR photocycle constituted of several intermediary states that differ in retinal conformation, protein backbone conformation and protonation state of specific groups, . Although this protein has been widely studied, many aspects of the functional mechanisms of ChR are far from understood on the molecular level. There are still many questions open, such as the identification of the amino acids that change their protonation state during the photocycle, or a deep understanding of the role of water molecules in the ion channel when the pore that allows the flow of ions is open [1]. In order to address these issues, a spectroscopic approach is required, since it is the only technique able to provide information at the single chemical bond level. However, as already mentioned, the well- established IR spectroscopy is usually performed on large amounts of purified light-sensitive proteins as the IR signal strength deriving from the conformational changes of proteins under absorption of visible light is very low. The aim of my research project is to investigate the light-induced conformational changes at the nanoscale using the experimental setup of IR nanospectroscopy already present in the laboratory at CLNS where I am carrying out my PhD.
The relevance of performing IR spectroscopy of light-sensitive TMPs, in particular of ChR, at the nanoscale is dual. From one hand it would allow to investigate the conformational changes of these protein receptors when embedded in their native cell membranes. This would have a biological relevance since the photocycle and the functions of these TMPs may be altered by the process of protein purification and embedding into artificial lipid membranes, a step that is necessary in the case of standard IR spectroscopy (where samples need to be extended over several millimeters). Moreover, this approach would allow to avoid the protein purification that is a quite sophisticated and expensive process . On the other hand, the possibility to perform IR spectroscopy of ChR at the nanoscale would open the way towards possible correlation studies between spectroscopy and patch clamp approach. The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of native cell membrane [2]. It has been extensively applied to the investigation of light-sensitive TMPs since it enables to retrieve information on their functions from the measure of the result of their light-activated photocycle. Patch-clamp technique enables to probe the ionic current down to the single ion channel level when these receptors are embedded in their native membranes. However, this approach does not provide information of the light-induced conformational changes of the proteins, therefore limiting the range of open questions that can be addressed. Up to now, IR spectroscopy and patch clamp have been applied to different samples since in the case of standard IR spectroscopy it is impossible to probe proteins embedded in their native cell membrane. However, it would be extremely important to combine these two techniques since they provide complementary information, i.e. protein conformational changes and ionic current, that is the result of the protein conformational changes.
[1] Lórenz-Fonfría, Víctor A., et al. Proceedings of the National Academy of Sciences 112.43 (2015): E5796-E5804
[2] Hamill, Owen P., et al. Pflügers Archiv 391.2 (1981): 85-100