Mechanical unfolding reveals stable 3-helix intermediates in talin and α-catenin
Fig 2
Unfolding force profiles of the studied protein constructs in constant velocity SMD.
(a) Unfolding forces for 5-helix talin rod R9 and R11 have similar profiles and show two peaks, which correspond to breaking of 5-helix and 3-helix state respectively. (b) R9 constructs with disulphide clamps have similar force profiles to wild-type R9, but unfolding of 3-helix state was blocked. Tandem constructs for (c & d) R9 and (f) α-catenin (MI-MII) were unfolded through 3-helix state for both monomers simultaneously. (c) R9 (wt)–R9 (wt) tandem showed four peaks, corresponding to breaking of the 5h & 5h→3h (peak I), 5h→3h & 3h (peak II), 3h & 3h→0h (peak III), and 3h→0h & 0h (peak IV) respectively. (d) Tandems with the clamped 3-helix state in one monomer showed three peaks, lacking the peak for unfolding of the clamped 3-helix state. (e) Unfolding force for 4-helix R3 bundle has one peak that corresponds to collapsing of 3-helix state. (f) 4-helix α-catenin showed one peak for breaking the 3-helix state in each domain. Cysteine residues in (d) R9 tandems, that form disulphide clamps shown as magenta spheres. Structure snapshots correspond to force peaks highlighted with red dots.