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

Abstract

The giant muscle protein titin plays important roles in heart function. Mutations in titin have emerged as a major cause of familial cardiomyopathy. Missense mutations have been identified in cardiomyopathy patients; however, it is challenging to distinguish disease-causing mutations from benign ones. Given the importance of titin mechanics in heart function, it is critically important to elucidate the mechano-phenotypes of cardiomyopathy-causing mutations found in the elastic I-band part of cardiac titin. Using single-molecule atomic force microscopy (AFM) and equilibrium chemical denaturation, we investigated the mechanical and thermodynamic effects of two missense mutations, R57C-I94 and S22P-I84, found in the elastic I-band part of cardiac titin that were predicted to be likely causing cardiomyopathy by bioinformatics analysis. Our AFM results showed that mutation R57C had a significant destabilization effect on the I94 module. R57C reduced the mechanical unfolding force of I94 by ∼30–40 pN, accelerated the unfolding kinetics, and decelerated the folding. These effects collectively increased the unfolding propensity of I94, likely resulting in altered titin elasticity. In comparison, S22P led to only modest destabilization of I84, with a decrease in unfolding force by ∼10 pN. It is unlikely that such a modest destabilization would lead to a change in titin elasticity. These results will serve as the first step toward elucidating mechano-phenotypes of cardiomyopathy-causing mutations in the elastic I-band.

De novo design of knotted tandem repeat proteins

“…The tensile strength of this construct is considerably higher than other known purely alpha-helical domains such as spectrin (around 40–50 pN) 25 . However, the measured unfolding force is weaker than other naturally occurring proteins such as muscle protein Titin IG27 (around 250 pN) 26 , as well as other de novo designed proteins such as Top7 (approximately 150 pN) 27 , or the ultrastable pulling handle ClfA (around 2400 pN) 28 at similar force loading rates. Those constructs derive their strength from sheared, paired beta strands.…”

Heterogeneity in conformational state space enhances the force-tolerance of mechanosensory proteins

“…Serine to proline isn't unique for Cdh23, relatively abundant with phenotypes 36 . Even for mechanosensitive Titin proteins, Ser22 to Pro mutation is reported with a phenotype of cardiomyopathy 37 .…”

Viscoelasticity of single folded proteins using dynamic atomic force microscopy

“…In this sense, dynamic AFM serves as a tool to identify the actual alteration in mechanical properties, such as elasticity and viscosity, due to mutation. Early hearing loss 173,174 and cardiomyopathy 175 are two of the examples from arguably a long list of diseases which are suspected to be a result of malfunctioning of mechano-sensitive proteins due to altered mechanical properties.…”