Single-Molecule Force Spectroscopy Studies of Missense Titin Mutations That Are Likely Causing Cardiomyopathy

Figures

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  Figure 1. (A) Schematics of cardiac titin N2B and N2BA isoforms. Blue asterisks indicate the locations of the missense mutations identified in cardiac titin N2B and N2BA isoforms. (B) Missense mutations identified in titin N2B and N2BA isoforms that are possibly and likely causing cardiomyopathy.

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  Figure 2. Three-dimensional structures of (A) I94, (B) R57C-I94, (C) I84, and (D) S22P-I84 predicted by homology modeling. Both I94 and I84 modules fold into a β-sandwich structure, similar to that of I91. Homology modeling also predicted that mutations R57C and S22P do not alter the three-dimensional structure of I94 and I84 significantly.

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  Figure 3. Equilibrium denaturation curves of I94, (A) I94-R57C, and I84, (B) I84-S22P. Solid lines are fits of the experimental data to a two-state unfolding–folding model. The fitting parameters are listed in Table S1.

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  Figure 4. Mechanical properties of I94 and R57C-I94. (A) Schematics of the single-molecule AFM pulling experiments using Coh-Doc interactions. Representative force–extension curves of (B) (GB1-I94)4 and (C) (GB1-R57C–I94)4. Two groups of unfolding force events were observed. Unfolding events colored black display a contour length increment ΔLc of 18 nm and correspond to the unfolding of GB1 domains, while unfolding events colored red display a contour length increment ΔLc of 28 nm and correspond to the unfolding of I94 or R57C-I94 domains. (D) Unfolding force histograms of I94 and R57C-I94 at a pulling speed of 400 nm/s. The average unfolding force is 187 ± 33 pN (avg. ± SD) for wt I94 (n = 208) or 145 ± 32 pN for R57C-I94 (n = 291). (E) Pulling speed dependence of the unfolding forces. Solid lines are Monte Carlo simulation results using a Δxu of 0.17 nm and α0 values of 0.025 s–1 for wt I94 and 0.08 s–1 for R57C-I94. The error bars are standard errors of the mean (sem).

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  Figure 5. Mechanical properties of I84 and S22P-I84. Representative force–extension curves of (A) (GB1-I84)4 and (B) (GB1-S22P-I84)4. Two groups of unfolding force events were observed. Unfolding events colored black display a contour length increment ΔLc of 18 nm and correspond to the unfolding of GB1 domains, while unfolding events colored red display a contour length increment ΔLc of 28 nm and correspond to the unfolding of I84 or S22P-I84 domains. (C) Unfolding force histograms of I84 and S22P-I84 at a pulling speed of 400 nm/s. The average unfolding force is 180 ± 30 pN (avg. ± SD) for wt I84 (n = 276) or 167 ± 30 pN for S22P-I84 (n = 232). (D) Pulling speed dependence of the unfolding forces. Solid lines are Monte Carlo simulation results using a Δxu of 0.23 nm and α0 values of 0.002 s–1 for wt I84 and 0.004 s–1 for S22P-I84. Errors bars correspond to the sem.

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  Figure 6. Effects of mutations on the folding kinetics of I94 and I84. (A) Schematics of the double-pulse AFM protocol in measuring the folding kinetics of proteins. (B) Representative refolding curves of full length (GB1-I94)4. In the first pulse, four GB1 and four I94 unfolding events were observed. In the second pulse, four GB1 unfolding events were observed due to its fast-folding kinetics, while the number of refolded I94 depended on Δt. The refolding experiments were carried out in the presence of 5 mM dithiothreitol, which prevented endogenous cysteine residues in I94 from forming a disulfide bond or reacting with unreacted maleimide. Plots of folding probability vs Δt for (C) I94 and R57C-I94 and (D) I84 and S22P-I84. Solid lines are the fitting to the first-order rate law Pf = 1 – exp(−β0t).