Supplementary Figure 2: Spatial vicinity between protons in αB, as observed by RFDR MAS NMR spectroscopy.
From: The chaperone αB-crystallin uses different interfaces to capture an amorphous and an amyloid client
(a) Section of the 1H-15N correlation MAS NMR spectrum of perdeuterated αB (black) showing the two cross-peaks for I133 (A and B). The corresponding 2D strips of the 3D hCXhNH spectra (red) demonstrate the degeneracy in the 13C dimension for both states and thus the benefits of introducing the 1H dimension to resolve the two structural states. 2D strips of the 3D 1H-1H RFDR spectrum (green) show the spatial connectivities between the backbone amides of I133-A and -B and the neighboring loop residues (including side-chain hydroxyl groups). The asterisks denote diagonal peaks. Both signals of I133 are devoid of exchange peaks with bulk water (4.7 ppm), which is in contrast e.g. to the more solvent-accessible loop residues S85–L89 (see panel f). (b) The asymmetric dimer interface II in the structural model of Braun, N. et al., Proc. Natl. Acad. Sci. U. S. A. 108, 20491–20496, 2011, and the different locations of residue I133 (magenta). The two protomers are colored in white (extended conformer) and green (bent conformer), respectively. The two-fold point-symmetry of the ACD dimer is disturbed by the differential arrangement of the corresponding NTDext/CTDext and NTDbent/CTDbent, respectively. (c) Close-up view of the loop residues preceeding I133 in the dimer structure of αB (Jehle, S. et al., Nat. Struct. Mol. Biol. 17, 1037–1042, 2010; Braun, N. et al., Proc. Natl. Acad. Sci. U. S. A. 108, 20491–20496, 2011). The spatial vicinity between the amide protons of I133, T132, L131 and D129 is confirmed by the observed RFDR contacts in (a). (d) Different register of the dimer interface I as observed in different X-ray structures of the excised αB dimer (Bagnéris, C. et al., J. Mol. Biol. 392, 1242–1252, 2009; Laganowsky, A. et al., Protein Sci. 19, 1031–1043, 2010). The interfaces are termed API and APII register and have the point-symmetric axis centered at residue F118 and E117, respectively. (e) 2D strips extracted from the 3D 1H-1H RFDR MAS NMR spectrum of αB. The strip plot shows inter-strand correlations between residues S115 and H119 (β6+7) supporting an APII register in full-length αB as indicated in the red box in (d). (f) Spatial vicinity between loop residues S85–L89. 2D strips extracted from 3D 1H-1H RFDR MAS NMR spectra recorded at 40 kHz MAS. Spatial contacts are indicated by dashed lines to guide the eye. Asterisks denote diagonal peaks. (g) Structure of the loop connecting strands β3 and β4 (residues S85–L89) in αB according to Braun, N. et al., Proc. Natl. Acad. Sci. U. S. A. 108, 20491–20496, 2011. (h) Imidazole region of 2D 1H-15N correlation spectra recorded on full-length αB (CP-MAS, red) and αB10m (solution-state HMQC, black). The two resonances of H104 (A and B) reveal a comparable signal pattern in strips of the 3D 1H-1H RFDR spectrum in (i). The spatial vicinity to the backbone amide of R116 represents a strand-strand contact involving β5 and β6+7. (j) Residues close to the side-chain of H104 in the structural model of αB according to Braun, N. et al., Proc. Natl. Acad. Sci. U. S. A. 108, 20491–20496, 2011.
