Unfolding patterns of tandem α-helical bundles as determined by smAFM.

<p>(a-d) Talin R9 (wt)–R9 (wt) (47 traces). (e-h) R9 (N/C-clamps)–R9 (wt) (34 traces). (i-l) α-catenin (M_I-M_II) (40 traces). (a, e & i) length histogram indicating the distance from unfolding event to HaloTag standard; (b, f & j) scatter plot showing force vs position for all unfolding events; histogram the force associated with unfolding events; (d,h & l) representative force extension retraction curve (blue, cyan & black line) showing how the unfolding events relate to scatter plot and length histogram (black dotted line), gray line represents approach curve for AFM tip. Gray dotted lines on (c, g & k) histograms represent Gaussian fits.</p

Schematic representation of talin rod unfolding through stable 3-helix intermediates.

<p>Without force, the talin rod subdomains remain intact and no VBSs are exposed. Under low load weak 4-helix bundles unfold to stable 3-helix intermediates. As the force increases, some of the 5-helix bundles unfold, forming the 3-helix intermediates along talin rod structure. Force-regulated unfolding of the talin rod changes affinity to different binding partners. RIAM and DLC1 are known to bind folded bundles, while recruitment of vinculin requires partial or complete unfolding of rod domains. VBSs that are located at terminal helices of the R2, R6, R7, and R10 bundles become available after unfolding to the 3-helix state (panel “Low force”).</p

Unfolding force profiles of the studied protein constructs in constant velocity SMD.

<p>(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 (M_I-M_II) 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.</p

α-helix bundle mechanical stability.

<p>(a) Schematic representation of talin rod bundles and (b) α-catenin domains. Vinculin binding sites colored in blue. (c) Cartoon illustration of R9, where Cα of N-terminal residue colored in blue and Cα of C-terminal residue is red. (d) SMD water box and (e) smAFMsetup, that were used for end-to-end stretching of studied proteins. Unfolding force profiles of talin rod R9 in (f) SMD and (g) smAFM experiments, where after collapsing of 5-helix bundle (peak I) the stable 3-helix intermediate was found (peak II). α-helix bundles used in unfolding experiments highlighted with yellow.</p

Unfolding patterns of the α-helix monomers as determined by smAFM.

<p>(a-d) R9 (87 traces). (e-h) R11 (79 traces). (a & e) length histogram indicating the distance from unfolding event to HaloTag standard; (b & f) scatter plot showing force vs position for all unfolding events; (c & g) histogram the force associated with unfolding events; (d & h) representative force extension retraction curve (green & magenta line) showing how the unfolding events relate to scatter plot and length histogram (black dotted line), gray line represents approach curve for AFM tip. Gray dotted lines on (c & g) histograms represent Gaussian fits.</p

Representative structure snapshots from SMD.

<p>Talin rod domain bundles and α-catenin (M_I-M_II) in constant velocity SMD simulations. Structures were taken (a) at 0, 5, 10 and 15 ns for monomers, and (b) at 0, 10, 20 and 30 ns for tandem constructs. Cysteine residues forming the disulphide clamps shown as magenta spheres. Unfolded structures are cut away and presented by dashed line with a helix identifier.</p

Stability of the intermediate states.

<p>Talin rod (a) R3 and (b) R9 over time in constant force SMD using constant force pulling at different force regimes. R9 is a strong 5-helix bundle, while stable 3-helix intermediates were observed in both R3 and R9 (gray shading). The pink trace in panel (b) corresponds to end-to-end distance measured under 200 pN applied force, where 220 pN simulation was used as a starting structure after passing the 5h to 3h transition (dashed gray arrow).</p