FIGURE 5.
Size exclusion chromatography of wild-type aCRY, aCRYΔCCT and the C482A mutant at injected concentrations of 0.5 mm. A, samples were illuminated for 10 s with 451 nm to convert FADox to FADH•, which was selectively detected at 630 nm. Wild-type aCRY and the C482A mutant eluted at 13.2 ml, which corresponds to an apparent molecular mass of 130 kDa. Considering a theoretical molecular mass of 66.4 kDa of aCRY, the bands can be assigned to dimers. aCRYΔCCT predominantly eluted at 14.8 ml, which corresponds to an apparent molecular mass of 60 kDa and can accordingly be assigned to a monomer. B, samples were illuminated for 45 s with 451 nm and 630 nm to convert FADox to FADH• and FADH−, which were detected at 370 nm. Wild-type aCRY shows an additional, small peak at 11.4 ml (314 kDa), which indicates a tetrameric species. This light-induced oligomerization can be abolished by the addition of the reducing agent TCEP. C, samples were treated as in B. In the C482A mutant, light-induced oligomerization is not observed. In contrast, aCRYΔCCT shows an additional peak at 13.3 ml, which can be assigned to a dimeric species. Therefore, some light-induced oligomerization was found for both wild-type aCRY and aCRYΔCCT.