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.
Highlights
A mutation found in titin has been linked to arrhythmogenic cardiomyopathy (AC)
Previous single molecule force spectroscopy and molecular dynamics studies have shown that residues in the AЈB loop are crucial for determining the structural stability of Ig domains via non-covalent interactions with residues in the G -strand [21,22], with AЈ mutations either increasing or decreasing the force needed to unfold the domain [23]
The last force peak is due to the fully unfolded Ig10 5-mer polypeptide displacing from the cantilever tip
Summary
A mutation found in titin has been linked to arrhythmogenic cardiomyopathy (AC). Results: The mutation increases Ig10 instability and susceptibility to degradation. 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. The recently discovered AC-linked titin mutation [4] is found in the tenth Ig domain (Ig10) of titin’s I-band region. Exon 37 encodes for the tenth Ig domain in the proximal tandem Ig segment of the I-band, and the mutation changes the native threonine residue into isoleucine. Previous single molecule force spectroscopy and molecular dynamics studies have shown that residues in the AЈB loop are crucial for determining the structural stability of Ig domains via non-covalent interactions with residues in the G -strand [21,22], with AЈ mutations either increasing or decreasing the force needed to unfold the domain [23]. A critical first step toward elucidating the relationship between mutated Ig10 and arrhythmogenic cardiomyopathies is determination of the functional effect of the T16I mutation at the single molecule level
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