Computational and single-molecule force studies of a macro domain protein reveal a key molecular determinant for mechanical stability

Abstract

Resolving molecular determinants of mechanical stability of proteins is crucial in the rational design of advanced biomaterials for use in biomedical and nanotechnological applications. Here we present an interdisciplinary study combining bioinformatics screening, steered molecular dynamics simulations, protein engineering, and single-molecule force spectroscopy that explores the mechanical properties of a macro domain protein with mixed α + β topology. The unique architecture is defined by a single seven-stranded β-sheet in the core of the protein flanked by five α-helices. Unlike mechanically stable proteins studied thus far, the macro domain provides the distinct advantage of having the key load-bearing hydrogen bonds (H bonds) buried in the hydrophobic core protected from water attacks. This feature allows direct measurement of the force required to break apart the load-bearing H bonds under locally hydrophobic conditions. Steered molecular dynamics simulations predicted extremely high mechanical stability of the macro domain by using constant velocity and constant force methods. Single-molecule force spectroscopy experiments confirm the exceptional mechanical strength of the macro domain, measuring a rupture force as high as 570 pN. Furthermore, through selective deletion of shielding peptide segments, we examined the same key H bonds under hydrophilic environments in which the β-strands are exposed to solvent and verify that the high mechanical stability of the macro domain results from excellent shielding of the load-bearing H bonds from competing water. Our study reveals that shielding water accessibility to the load-bearing strands is a critical molecular determinant for enhancing the mechanical stability of proteins.