S. aureus MscL Is a Pentamer In Vivo but of Variable Stoichiometries In Vitro: Implications for Detergent-Solubilized Membrane Proteins
Figure 4
LDAO stabilizes a MscL tetrameric oligomeric state as measured by crosslinking, SEC-MALS, and Sedimentation Velocity Centrifugation.
(A) Western blot analysis of DSS-treated SaMscL solubilized with either Triton-X100 or LDAO yield different quantities of tetramers and pentamers. (B) A representative SEC-MALS experiment of SaMscL in LDAO is shown with the UV chromatogram colored blue and the 90° light scattering chromatogram colored red. The total, SaMscL, and LDAO masses are shown as black, green, and orange lines across the elution peak, respectively. The mass of the SaMscL monomer is 14.4 kDa and the tetramer mass is 57.8 kDa. (C) SaMscL in 4 mM LDAO was subjected to sedimentation velocity centrifugation at 50,000 rpm, and the data were analyzed using the noninteracting “species model” of SEDPHAT. Four species, including the detergent micelles, were analyzed. The molar mass of the dominant sedimenting species was 62.5 kDa. In the upper part, the individual data points are depicted as circles, and the best-fit model to those data is shown as lines. The data and fit lines are color coded by color: Violet for the earliest scans, then progressing through indigo, blue, green, yellow, orange, and red as the scans go further forward in time. For clarity only every other scan used in the data analysis is shown. In the lower part, the residuals between the data points and the fitted line are shown and color coded as above.