Figure 1.

Purification of green opsin by size exclusion chromatography and analysis of glycosylation by SDS-PAGE and immunoblotting. Panel A is a size exclusion chromatogram as obtained from numerous purifications. It reveals that aggregates and oligomers eluted in fraction 11 whereas dimeric opsin eluted in fractions 12–18 (see also Figure S1). The 1D4 peptide used for affinity chromatography eluted in fraction 25. Panel B shows the absorbance measurements of fraction 15. The peak at 530 nm originates from the chromophore bound to green opsin; the absorbance at 363 nm is due to free retinal in the buffer, and the 280 nm absorbance results from aromatic amino acid residues Trp, Tyr, and Phe. These absorbance spectra are routinely applied to verify the purity and yield of the expressed pigment. Panel C shows Coomassie staining of glycosylated (lane 2) and deglycosylated (lane 3) opsin. Following PNGase F treatment, the deglycosylated receptor undergoes a mobility shift toward a molecular weight lower than that of the glycosylated receptor. Additionally, lane 3 reveals a thin band attributed to PNGase F at ∼32 kDa. Panel D displays immunoblotting analysis of the glycosylated (lane 2) and deglycosylated (lane 3) opsin. The monomer (30 kDa), dimer (60 kDa), tetramer (120 kDa), and higher oligomeric species can be detected by both SDS-PAGE and immunoblotting analyses. Lanes 1 and 4 in both panels contain protein markers. Panel E displays triplicate differential scanning fluorescence measurements of green opsin and bovine rhodopsin. The normalized fluorescence of green opsin (green line) was fitted to the Boltzmann equation (R = 0.9998, and R_sqr = 0.9997) that resulted in a melting temperature of 48 ± 0.06 °C. The corresponding temperature obtained for bovine rhodopsin (red line) (R = 0.9988, and R_sqr = 0.9976) revealed a melting temperature of 72 ± 0.28 °C. The difference between the two melting temperatures is 24 ± 0.29 °C.