Large-Scale Modulation of Titin Elasticity by S-Glutathionylation of Cryptic Cysteines

Abstract

The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole, and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric oxide signaling cascade that increases cardiac muscle elasticity. However, it is unknown whether S-glutathionylation regulates the elasticity of titin and cardiac tissue. Here, we use homology modeling techniques to show that most immunoglobulin (Ig) domains in the elastic I-band of titin contain cryptic cysteines, which are potential targets of S-glutathionylation triggered by physiological mechanical protein unfolding in the heart. We choose I91 as a representative Ig domain of titin to investigate the effects of S-glutathionylation in the elasticity of the protein. Using single-molecule force-clamp spectroscopy, we demonstrate that mechanical unfolding of I91 exposes two buried cysteine residues, which then can be S-glutathionylated by oxidized glutathione in the solution. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of I91, which unfolds at a rate two orders of magnitude faster following S-glutathionylation. In addition, S-glutathionylation severely compromises the ability of I91 to fold. Both effects, which are fully reversed by the enzyme glutaredoxin, soften the I91 domain. When extrapolated to all the Ig domains in the I-band, our observations predict that S-glutathionylation can trigger a highly extensible state of titin. Indeed, we show that S-glutathionylation of cryptic cysteines in titin mediates mechano-chemical modulation of the elasticity of human cardiomyocytes. Monte Carlo simulations illustrate that large-scale regulation of the elasticity of titin through posttranslational modification of cryptic cysteines can be achieved on time scales of minutes to hours. We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.