Structure of rhodopsin in monolayers at the air-water interface: a PM-IRRAS and X-ray reflectivity study.

Monomolecular films of the membrane protein rhodopsin have been investigated in situ at the air-water interface by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and X-ray reflectivity in order to find conditions that retain the protein secondary structure. The spreading of rhodopsin at 0 or 5 mN m(-1) followed by a 30 min incubation time at 21 degrees C resulted in the unfolding of rhodopsin, as evidenced from the large increase of its molecular area, its small monolayer thickness, and the extensive formation of beta-sheets at the expense of the alpha-helices originally present in rhodopsin. In contrast, when spreading is performed at 5 or 10 mN m(-1) followed by an immediate compression at, respectively, 4 or 21 degrees C, the secondary structure of rhodopsin is retained, and the thickness of these films is in good agreement with the size of rhodopsin determined from its crystal structure. The amide I/amide II ratio also allowed to determine that the orientation of rhodopsin only slightly changes with surface pressure and it remains almost unchanged when the film is maintained at 20 mN m(-1) for 120 min at 4 degrees C. In addition, the PM-IRRAS spectra of rod outer segment disk membranes in monolayers suggest that rhodopsin also retained its secondary structure in these films.