Trapping metarhodopsin I in 2D crystals
2D crystals of bovine rhodopsin with p22121 plane-group symmetry were obtained by dialysis of detergent-solubilized rhodopsin isolated from bovine rod outer segment membranes (6, 7). Addition of cholesterol prior to dialysis increases the yield of rhodopsin crystals, allowing the preparation of 5-10 mg amounts of 2D crystals with minimal protein aggregates for spectroscopic characterisation (8).
To trap meta I, the sample temperature was kept below 4 °C during all steps of the experiment. A spectrum of the crystals recorded before illumination shows an absorption peak at 498 nm, characteristic of the ground-state (black line). Spectra recorded immediately after illumination show a shift in the absorption maximum to 480 nm, characteristic of the formation of meta I (red line). Four cryoelectron microscopy grids were prepared from aliquots taken from the photometer cell, thus freeze-trapping meta I for subsequent structural studies. Preparation and manipulation of four grids typically took 20-30 minutes, after which a spectrum was recorded, to check that the sample had not changed significantly (blue line). The sample was then acid denatured, and a further spectrum was recorded (green line). The shift in absorption maximum to 470 nm, and absence of a peak at 380 nm, indicates that the retinal chromophore is still attached to the protein by a protonated Schiff base linkage (9). Fourier-transform infrared (FTIR) difference spectroscopy shows that bathorhodopsin, lumirhodopsin and meta I photointermediates formed in 2D crystals are very similar to those obtained from rhodopsin in native disk membranes (5). Further UV/Visible spectroscopy experiments showed that the photostationary state consisted of 60-70% meta I and 30-40% of cis photoproducts. The high meta I content in the 2D crystals, and its similarity to native meta I, made this an ideal photointermediate to characterise structurally.