Figure 4. SAS-5 forms higher-order assemblies in solution.
(A) Overlay of SEC-MALS chromatograms of MsyB-tagged SAS-5FLEX in multiple concentrations and (B) plot of average molecular weight from SEC-MALS analysis as a function of on-column protein concentration. The apparent Kd and molecular sizes of dimers, tetramers and hexamers are indicated. SAS-5FLEX-MsyB reaches an equimolar hexamer to tetramer ratio at the highest concentration we could assess. Fitting the experimental data under the assumption of ultimate hexamer formation yielded Kd values comparable to those of the SAS-5 coiled coil in isolation (compared B with Figure 2H). (C,D) Similar SEC-MALS analysis of MsyB-tagged SAS-52–265. SAS-52–265-MsyB reaches a 3:1 hexamer to tetramer ratio at the highest concentration point. (E) Native mass-spectrometry electrospray ionization spectrum of a 20 μM sample of SAS-52–265, showing relative abundance of protein species as a function of mass to charge ratio. The charged states and protein oligomeric forms corresponding to specific peaks are indicated. Odd-numbered oligomeric forms (monomers, trimers etc) likely correspond to in-flight breakdown of higher- order protein assemblies.