Fig. 4.

bR-BE₂ unfolded structure probed by pulse proteolysis assay. (A) Time course of subtilisin digestion of folded bR-BE₂ in the presence of WT-mSA. In the pathway to the unfolded state, the protein becomes doubly bound in the native state, followed by slower unfolding of this strained folded state. Thus, to observe proteolysis in the native state, digestions were started 10 min after the addition of WT-mSA, before the protein had time to unfold. The band corresponding to bR-BE₂ with two bound mSAs is highlighted by the red box. The band is still present after 15 min of digestion. (B) Time course of subtilisin digestion of steric trap unfolded bR-BE₂. Digestions were started 1 d after the addition of WT-mSA to allow time to unfold. The steric trapped unfolded bR-BE₂ was digested completely within 3–5 min.