Supplementary Figure 3: Differential behavior of N- and C-terminal regions in αB.

(a) Accessibility of N- and C-terminal proteolytic cleavage-sites in αB. SDS-PAGE of His₆-Ek–αB (25 kDa, where Ek denotes the recognition site of the protease enterokinase) before (lane 1), after 15 h (lane 2) and after 40 h (lane 3) incubation with enterokinase at 4 °C. Molecular weights of relevant marker bands (M) are indicated. The asterisks label the non-specific by-products, which were analyzed by (b) MALDI-TOF MS of the protein solution after enterokinase degradation and (c) MALDI-TOF/TOF MS² of the corresponding gel bands of αB (blue) and αB* (red) after tryptic digest. The MS results clearly identify the by-product as a truncation variant missing the C-terminal residues T158–K175 (1,943 Da). (d) Enlarged view of the αB 24-mer⁵ showing three bent conformers (blue) and three extended conformers (red) participating in the intermolecular packing of the six NTDs at the so-called “closed” three-fold point-symmetric axis (C3_closed). The “open” three-fold point-symmetric axis (C3_open) centered in the hexameric ring is also indicated. The locations of the N- and C-termini of bent and extended conformers are highlighted to visualize the different accessibilities of the termini: the bent conformer exposes both termini towards the surface of the oligomer, whereas the extended conformer locates both termini close to each other in the interior of the assembly. This asymmetric arrangement is supported by the occurrence of specific and non-specific processing at the dissimilar N- and C-termini, respectively. (e) Diffusion attenuation profiles for the major (black) and minor conformers (red) of the αB CTD are plotted for residues R157, A172 and K175. In all cases, both conformers decay equally. The average translational diffusion coefficient amounts to 2.65 (± 0.16) × 10⁻¹¹ m²/s. The profiles were recorded at 295 K and an external magnetic field of 21.1 T. (f) Simulation of attenuation profiles (dashed lines) for various αB n-mers as indicated in the legend. Data points of the experimental profiles are shown for both conformers of K175.