Analytical ultracentrifugation sedimentation velocity for the characterization of detergent-solubilized membrane proteins Ca++-ATPase and ExbB.
We have investigated the potential of new methods of analysis of sedimentation velocity (SV) analytical ultracentrifugation (AUC) for the characterization of detergent-solubilized membrane proteins. We analyze the membrane proteins Ca(++)-ATPase and ExbB solubilized with DDM (dodecyl-beta-D: -maltoside). SV is extremely well suited for characterizing sample heterogeneity. DDM micelles (s(20w) = 3.1 S) and complexes (Ca(++)-ATPase: s(20w) = 7.3 S; ExbB: s(20w) = 4 S) are easily distinguished. Using different detergent and protein concentrations, SV does not detect any evidence of self-association for the two proteins. An estimate of bound detergent of 0.9 g/g for Ca(++)-ATPase and 1.5 g/g for ExbB is obtained from the combined analysis of SV profiles obtained using absorbance and interference optics. Combining s(20w) with values of the hydrodynamic radius, R(s) = 5.5 nm for Ca(++)-ATPase or R(s) = 3.4 nm for ExbB, allows the determination of buoyant molar masses, M(b). In view of their M(b) and composition, Ca(++)-ATPase and ExbB are monomers in our experimental conditions. We conclude that one of the main advantages of SV versus other techniques is the possibility to ascertain the homogeneity of the samples and to focus on a given complex even in the presence of other impurities or aggregates. The relative rapidity of SV measurements also allows experiments on unstable samples.
Salvay
Andrés G
AG
CNRS, IBS, Laboratoire de Biophysique Moléculaire, Grenoble 38027, France.
Santamaria
Monica
M
le Maire
Marc
M
Ebel
Christine
C