Posttranslational Modifications as Modulators of Mechanical Protein Folding

Abstract

Understanding the molecular basis of muscle elasticity is an important challenge in medicine. For example, mutations in the giant elastic protein titin are implicated in the etiology of dilated cardiomyopathies. We use single molecule force-clamp spectroscopy to study the folding and unfolding dynamics of single polyproteins composed of tandem repeats of titin Ig domains. Protein elasticity is due to the diffusional extension and collapse of a polyprotein along its free energy landscape. Using single molecule force-clamp traces we calculate the free energy of a polyprotein along its stretching coordinate under different mechanical force regimes. At high stretching forces the free energy is dominated by changes in entropy as the molecule unfolds and extends. When the force is quenched, enthalpic interactions become important as the polyprotein slowly progresses from collapsed states to molten globules and then to the mechanically stable native state. We also show that while the polyprotein is in the extended state, cysteine residues that are normally buried become exposed to the solution where they can be post-translationally modified. We have found that S-glutathionylation of such cryptic cysteines greatly decreases the mechanical stability of an Ig domain as well as its ability to fold, favoring more extensible states in titin. Thus, the free energy landscape of a protein extending and folding under force is strongly modulated by the redox state of a cell. We propose that posttranslational modification of cryptic cysteine residues is a major regulatory pathway of tissue elasticity. Accurate predictions of elastic phenotypes in titin will be possible after we fully understand how cellular chemistry modulates the physics of a polypeptide extending under force.