Intrinsically Disordered Titin PEVK as a Molecular Velcro: Salt-Bridge Dynamics and Elasticity

Abstract

It is increasingly recognized that many proteins are intrinsically disordered and do not have a unique compact structure as those found in globular proteins. Titin is a giant modular protein (3-4 MDa) found in the muscle sarcomere that is comprised of both globular and disordered modules. The elastic titin PEVK segment, with tandem repeats of ∼28 residue modules, plays a major role in the passive tension of skeletal and heart tissues. We have proposed based on AFM studies of a cloned titin PEVK fragment, that salt-bridges play a central role in the elasticity of this PEVK polyelectrolyte. We have engineered a construct of 15 repeats of a single titin 28-residue PEVK module (human exon 172). The 50 kDa polyprotein shows well-resolved NMR spectra in dilute solution and in highly concentrated gels. Both chemical shifts and sequential NOE's indicate the presence of polyproline II helices. From long-range NOE's, we observed, for the first time, stable K to E salt-bridges with non-random pairings. Simulated annealing with NMR restraints yielded a manifold of plausible structures for an exon 172 trimer showing many salt-bridges. Steered molecular dynamics simulations (SMD) were done to study how the manifold of salt-bridges evolves during the stretching experiment. Repeated SMD simulations at slow velocity (0.0005 nm/ps) show force spectra consistent with experimental AFM force spectra of the polyprotein. SMD shows that salt-bridges occur even at high degrees of stretch and that these short range interactions are in integral part of the mechanical properties of PEVK. We propose that the long-range, non-stereospecific nature of electrostatic interactions provide a facile mechanism to tether and untether the flexible chains, which in turn affect elasticity as well as control the accessibility of protein-protein interaction to these nanogel-like proteins.