Mechanical unfolding reveals stable 3-helix intermediates in talin and ?-catenin.

ABSTRACT: Mechanical stability is a key feature in the regulation of structural scaffolding proteins and their functions. Despite the abundance of ?-helical structures among the human proteome and their undisputed importance in health and disease, the fundamental principles of their behavior under mechanical load are poorly understood. Talin and ?-catenin are two key molecules in focal adhesions and adherens junctions, respectively. In this study, we used a combination of atomistic steered molecular dynamics (SMD) simulations, polyprotein engineering, and single-molecule atomic force microscopy (smAFM) to investigate unfolding of these proteins. SMD simulations revealed that talin rod ?-helix bundles as well as ?-catenin ?-helix domains unfold through stable 3-helix intermediates. While the 5-helix bundles were found to be mechanically stable, a second stable conformation corresponding to the 3-helix state was revealed. Mechanically weaker 4-helix bundles easily unfolded into a stable 3-helix conformation. The results of smAFM experiments were in agreement with the findings of the computational simulations. The disulfide clamp mutants, designed to protect the stable state, support the 3-helix intermediate model in both experimental and computational setups. As a result, multiple discrete unfolding intermediate states in the talin and ?-catenin unfolding pathway were discovered. Better understanding of the mechanical unfolding mechanism of ?-helix proteins is a key step towards comprehensive models describing the mechanoregulation of proteins.

SUBMITTER: Mykuliak VV 

PROVIDER: S-EPMC5940241 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature