Single molecule force spectroscopy on titin implicates immunoglobulin domain stability as a cardiac disease mechanism

Abstract

Titin plays crucial roles in sarcomere organization and cardiac elasticity by acting as an intrasarcomeric molecular spring. A mutation in the tenth Ig-like domain of titin's spring region is associated with arrhythmogenic cardiomyopathy, a disease characterized by ventricular arrhythmias leading to cardiac arrest and sudden death. Titin is the first sarcomeric protein linked to arrhythmogenic cardiomyopathy. To characterize the disease mechanism, we have used atomic force microscopy to directly measure the effects that the disease-linked point mutation (T16I) has on the mechanical and kinetic stability of Ig10 at the single molecule level. The mutation decreases the force needed to unfold Ig10 and increases its rate of unfolding 4-fold. We also found that T16I Ig10 is more prone to degradation, presumably due to compromised local protein structure. Overall, the disease-linked mutation weakens the structural integrity of titin's Ig10 domain and suggests an Ig domain disease mechanism.

FIGURE 1.

A, schematic of a cardiac sarcomere. A single titin molecule spans from the Z-disk to the M-band and contains a large elastic I-band region consisting of tandem Ig domains, the N2B element, and PEVK sequence. B, schematic of part of titin's I-band region. The AC-linked point mutation is in the tenth proximal Ig domain. Note that the 15 proximal and 22 distal Ig domains of the N2B titin isoform are not all shown. C, Ig10 tertiary structure predicted from homology modeling (19). D, alignment of predicted Ig10 secondary structure (20) with solved titin Ig domains shows that the T16I mutation resides in the A′B peptide loop (40). Residues highlighted in blue have β-strand secondary structure (predicted in Ig10).

FIGURE 2.

A, simplified AFM schematic. B, representative force-extension traces of WT and T16I Ig10 5-mer stretched at 1000 nm/s. The red trace indicates the cantilever tip approaching the protein-coated slide surface, and the blue trace indicates the tip moving away from the surface with a tethered protein. The first five low force peaks are due to Ig domain unfolding, with an increase in contour length and subsequent decrease in force immediately following each unfolding event. The last force peak is due to the fully unfolded 5-mer being displaced from the cantilever tip.

FIGURE 3.

cdfs of unfolding forces for WT and T16I Ig10 5-mers. For each force value, the y axis value indicates the percentage of domains that unfolded at or below that force. The blue traces are the cdfs of peaks 1–4. The red traces are the cdfs of peaks 1–4 after transforming to the parent cdf using order statistics (30). The cdf of peak 5 is also in red (because we are stretching a 5-mer, by definition the cdf of the last unfolding peak forces is the parent cdf). The cyan traces are the average of the five parent cdf (red) traces. These averaged parent cdfs were used to compare experimental data with simulated data (after simulated data were similarly processed). This analysis reduces the spread in unfolding force data and allows all peaks to be analyzed together, which increases the sample size and improves fitting accuracy.

FIGURE 4.

AFM force clamp protocol. A, tethered proteins were held at constant force for 5 s. After an unfolding event (arrows), the cantilever quickly retracted from the surface until the tension level was restored, resulting in a stepwise pattern. The extension difference (ΔExt) between steps represents the extension increase following an unfolding event. The hold time prior to a domain unfolding is measured from when the residual tension is initially reached (start of the purple trace). For example, the second Ig domain takes t = Δt₁ + Δt₂ to unfold. B, histograms of extension increases. The histograms represent the compilation of all force clamp data. The red Gaussian trace is a best fit to the histogram. The smooth distribution is centered at 23.3 nm for WT Ig10 and 23.0 nm for T16I. The data that do not fit the Gaussian distribution represent the individual segments of two-step unfolding events. Two-step unfolding is 5 times more prevalent in WT Ig10.

FIGURE 5.

Force-dependent unfolding rates. From the collection of unfolding times acquired at a given residual tension, we are able to fit the cumulative distribution function to 1 − e^−α(F)·t, where α(F) is the unfolding rate at a given force. The empirical cdfs shown were generated from force clamp values between 63 and 67 pN (average ∼65 pN). The smooth fits yield α(F = 65) = 1.135 s⁻¹ for WT Ig10 and α(F = 65) = 6.865 s⁻¹ for T16I Ig10. Inset, force-dependent unfolding rates for WT and T16I Ig10. Error bars, S.E.

FIGURE 6.

AFM refolding protocol. A tethered molecule was unfolded and then relaxed to allow for domain refolding. Restretching the molecule and identification of overlapping force peaks allows determination of how many unfolded domains refolded while the molecule was relaxed. The blue trace is the initial stretch. After extension and force triggers were reached, the cantilever tip was driven back toward the surface (low force portion of the orange trace, from 110 to 25 nm) to release tension in the molecule. After holding for a set period of time, the molecule was restretched fully (orange trace from 25 to 140 nm).

FIGURE 7.

Ig10 proteolysis. A, SDS-polyacrylamide gel showing degradation products of WT and T16I Ig10 5-mer following 10-, 30-, and 90-min trypsin incubations. The arrowhead denotes the “primary degradation product” of T16I Ig10 alluded to under “Discussion.” WT Ig10 is relatively impervious to peptide bond cleavage. B, the amount of full-length 5-mer remaining as a function of trypsin incubation time ± S.E. (error bars). C, full sequence of the T16I Ig10 5-mer. The only difference between WT and T16I 5-mer is the Thr → Ile mutation in each Ig domain. Mass spectrometry found that the primary degradation product of T16I begins with DIPTTENLY in the N-terminal linker sequence and ends at Lys-17 in the last Ig domain.

FIGURE 8.

A, degradation assay with recombinant Ig7–13 (with and without T16I in Ig10) and heart extract. Ig7–13 is exposed to all of the proteases naturally found in the heart. The mutant Ig7–13 protein has a larger N-terminal linker sequence than its wild type counterpart and has mobility similar to that of a heart extract protein. These overlapping bands were fit with a double Gaussian to isolate the intensity due to full-length Ig7–13 mutant. This experiment was performed at pH ∼6.3 and ∼7.4, but no pH dependence was found. B, Ig7–13 with the T16I Ig10 domain degrades much more rapidly than WT Ig7–13 at both pH values, which indicates increased degradation rate. pH values used were 6.33 ± 0.06 (S.E.) and 7.38 ± 0.03 (S.E.); n = 3 for each bar. ***, p < 0.001 between WT and mutant using a two-way analysis of variance and Bonferroni post hoc test.

FIGURE 9.

In vivo unfolding simulations. The force experienced by titin's elastic I-band region in the beating heart was simulated and used to estimate the percentage of Ig domains that are unfolded in vivo. WT Ig10 is unfolded an order of magnitude more often than other Ig domains, which shows that Ig10 is less stable than other Ig domains even without the T16I mutation. T16I Ig10 is unfolded 2 orders of magnitude more than other Ig domains. Note the breaks in the y axis.

FIGURE 10.

Pulse proteolysis. A, the fraction of WT Ig10 monomer that remains folded decreases as urea concentration increases. The C_m value indicates the urea concentration where folded and unfolded protein levels are equal. B, the free energy of unfolding at various urea concentrations in the transition zone (where unfolded and folded populations are both substantial) is determined from the percentage of folded and unfolded Ig10; extrapolating to 0 m urea allows determination of the free energy of unfolding in the absence of any denaturant. Error bars, S.E.