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.
This case study illustrates the impact of this method as a powerful tool to study membrane proteins and their detergent belt by allowing accurate mass measurements without column calibration. Generally, classic calibration based on soluble proteins does not apply in the case of pdc, whose volume and shape also depend on the detergent fraction.
The method described in this article serves to identify the quaternary structure of membrane proteins and to follow the presence of the correct folding of oligomeric proteins throughout purification. When the expected quaternary structure is known, this elegant approach provides clues about the retention of the native and thus active protein structure without performing laborious activity tests. This biophysical characterization technique improves thus dramatically the probability of success when carrying out crystallization or other biochemical studies requiring active and homogeneous protein samples.