Superposition of the gel-filtration chromatogram (OD280 trace only) of purified CorA revealed that CorADDM eluted slightly earlier than CorALDAO and that the elution profile of the latter was sharper and more symmetrical. However, SEC/MALS/refractometry/UV allowed us to measure the masses of the whole pdc in DDM (355 kDa) as well as the respective masses of the protein fraction (241 kDa) and the detergent belt (114 kDa), as illustrated in Figure 2A.
The mass of the protein fraction nicely fits with the theoretical mass of the CorA pentamer (5 x 46.9 kDa = 234.5 kDa vs 241 kDa), i.e., the physiological quaternary structure. With LDAO, in contrast, we found a total pdc mass of 93 kDa, corresponding to the sum of 47 kDa of protein and 46 kDa of detergent.
Thus, LDAO led to complete dissociation of CorA into monomer-containing-micelles, a non-native state of this protein (Figure 2B). These results helped us distinguish which detergents are suitable for purifying this archeal CorA using ~100 μg of protein for each injection.