Fig. 4.

SdrG B2 and SdrD B1 are even stronger than SdrG B1 and have different Ca²⁺ affinities. a Equilibrated homology model of SdrG B2 (brown) with Ca²⁺ (yellow) binding loops one to three marked, coordinating amino-acid side chains shown as sticks (coloring as in Fig. 3a). b Equilibrated crystal structure of SdrD B1 (blue, PDB 4JDZ). c Cycling of SdrD B1 between 10 mM Ca²⁺ and 10 mM EDTA, a strong state in Ca²⁺ over 2000 pN emerges. The weak state is bimodal peaking at ~ 550 pN and ~ 850 pN (gray arrow), fit with a superposition of two BE p(F) functions (see methods). Very few unfolding events appear in 10 mM EDTA, thus the Ca²⁺-chelation must be sufficient to completely unfold most SdrD B1 domains. The strong state is also bimodal in low Ca²⁺ conditions see Supplementary Fig. 8. d Relative fraction of states in different buffers: 10 mM Ca²⁺, 10 mM EDTA, and 50 nM Ca²⁺ (applied after 10 mM EDTA) for SdrG B1, B2 and SdrD B1 compared with a single cantilever: no unfolding event (unfolded protein, gray), weak state (< 1000 pN, blue), strong state (> 1500 pN, red). Almost all SdrD B1 and SdrG B2 domains are unfolded in EDTA and only a fraction refolds in low Ca²⁺. SdrG B2 has the lowest affinity for Ca²⁺ as it shows the least folded events in low Ca²⁺. e Comparison of relative unfolding forces of all B domains at 1.6 µm s⁻¹ with a single cantilever. The strongest state of SdrD B1 (blue, dashed line) is the most mechanostable, followed by SdrG B2 (brown, dash-dotted line), then SdrG B1 (green, dotted line). The weak states of SdrD B1 and SdrG B2 are bimodal and best described with a superposition of two BE fits, whereas SdrG B1 only has a single weak state