Light-induced FTIR Spectroscopy of Visual Rhodopsin Microcrystals Grown in Lipidic Cubic Phase

• We applied light-induced FTIR spectroscopy to bovine rhodopsin microcrystals grown in lipidic cubic phase (LCP) to investigate the structural changes during photoreaction. • Comparison with native retinal membranes revealed both conserved and distinct features in hydrogen-bonding networks and helical rearrangements. • Notably, formation of Meta-I and Meta-IIa intermediates was observed in crystals, demonstrating that key activation steps proceed similarly in crystalline and native environments. • This work provides a crucial spectroscopic foundation for time-resolved structural studies using XFEL. Time-resolved X-ray crystallographic analysis of mammalian visual rhodopsin has allowed to visualize the cis -to- trans isomerization of the retinal chromophore, a pivotal event in the early stages of vision, in a temporal and atomic resolution. This achievement provides a foundation for visualizing the subsequent photoreaction dynamics of bovine rhodopsin, paving the way for a comprehensive understanding of the molecular mechanism underlying scotopic vision. However, a critical question remains: Do the structural changes induced by the photoreactions in crystalline environments faithfully mirror those occurring in native membrane environments? To start addressing this essential question and improve the reliability of future time-resolved X-ray crystallographic analyses, we first applied low-temperature light-induced FTIR spectroscopy to bovine rhodopsin microcrystals formed using the lipidic cubic phase (LCP) method, in order to compare with conformational changes in native retina membrane (rod outer segment). By encapsulating the microcrystals in custom-made FTIR measurement windows, we successfully obtained FTIR difference spectra of photoreaction intermediates trapped at various temperatures. A detailed comparison with spectra obtained from rhodopsin from native retina membrane revealed both similarities and differences in photoreaction-induced structural changes between crystalline and membrane environments, including alterations in local hydrogen-bonding networks and structural rearrangements of the α-helical backbone. Notably, the formation of Meta-I and Meta-IIa intermediates, which are precursors of G-protein activation, was confirmed even in the crystalline environment, demonstrating that the core photoreaction dynamics proceed comparably in both environments.