(A) All sHSPs are defined by a conserved α-crystallin domain (ACD), that is flanked by N-terminal and C-terminal regions (NTR and CTR, respectively). Constructs used in this study are shown and their quaternary structure (oligomer vs. dimer) is indicated. HSPB1 contains a single cysteine, located in the ACD, which was substituted with serine in NMR-PRE and HDXMS experiments across several constructs. Phosphorylation-mimicking mutations in the NTR along with mutation of the IXI motif in the CTR (to abrogate ACD-CTR interactions) yields a full-length dimer, used in HDXMS experiments here. The C-terminally truncated construct (NTR-ACD) was used predominantly for NMR assignments and PRE experiments. The N-terminally and C-terminally truncated B1-ACD construct was used for peptide-binding NMR experiments. (B) ACD homodimer structure (4MJH), with dotted line indicating axis of symmetry along which ACDs interact via β6+7 strands. Dotted arrows in the right panel indicate the axes of the dimer interface and β4/β8 grooves. (C) NTR alignment of better-characterized human sHSPs shows minimal sequence conservation aside from the ‘conserved’ region. The NTR sequence of HSPB1 was divided into six sub-regions for this study, which were probed in peptide form (with the exception of the highly hydrophobic insertion region). Sites of interest in this study are also indicated: 1) phosphorylation sites mutated to aspartate to mimic phosphorylation, 2) sites that were targeted for spin label attachment, and 3) three disease-associated mutations (G34R, P39L, and G84R).