Mechanically Unfolding the Small, Topologically Simple Protein L

Abstract

beta-sheet proteins are generally more able to resist mechanical deformation than alpha-helical proteins. Experiments measuring the mechanical resistance of beta-sheet proteins extended by their termini led to the hypothesis that parallel, directly hydrogen-bonded terminal beta-strands provide the greatest mechanical strength. Here we test this hypothesis by measuring the mechanical properties of protein L, a domain with a topology predicted to be mechanically strong, but with no known mechanical function. A pentamer of this small, topologically simple protein is resistant to mechanical deformation over a wide range of extension rates. Molecular dynamics simulations show the energy landscape for protein L is highly restricted for mechanical unfolding and that this protein unfolds by the shearing apart of two structural units in a mechanism similar to that proposed for ubiquitin, which belongs to the same structural class as protein L, but unfolds at a significantly higher force. These data suggest that the mechanism of mechanical unfolding is conserved in proteins within the same fold family and demonstrate that although the topology and presence of a hydrogen-bonded clamp are of central importance in determining mechanical strength, hydrophobic interactions also play an important role in modulating the mechanical resistance of these similar proteins.

Single-molecule force spectroscopy reveals a mechanically stable protein fold and the rational tuning of its mechanical stability

“…2D, red line). The ⌬x u for Top7 is similar to that of several other mechanically stable proteins (11,13,23). A small value of ⌬x u indicates that transition state for the mechanical unfolding of Top7 is structurally very similar to the native state.…”

“…These factors have made it difficult or impossible to predict and tune mechanical properties of proteins in a systematic and rational fashion. Consequently, the known mechanically stable proteins are restricted to only a limited number of protein folds, significantly limiting the potential exploration of elastomeric proteins for nanomechanical applications.A number of proteins of different topologies (6, 10) have been studied for their mechanical properties, and the importance of protein topology to the mechanical stability has emerged (6,(11)(12)(13). It is recognized that the vast majority of mechanically stable proteins identified so far share a common shear topology, in which the two terminal ␤ strands are arranged in parallel and are directly connected to each other by noncovalent interactions (6, 10).…”

“…It is recognized that the vast majority of mechanically stable proteins identified so far share a common shear topology, in which the two terminal ␤ strands are arranged in parallel and are directly connected to each other by noncovalent interactions (6, 10). The only known exceptions are green fluorescent protein (14), a tightly formed ␤-barrel protein, and ankyrin (15), a 22-24 repeat protein.Upon stretching of these mechanically stable proteins, the two terminal ␤ strands are sheared against each other and the noncovalent interactions connecting the two force-bearing strands provide resistance to mechanical unfolding (6,11,12,(16)(17)(18)(19)(20). These mechanically stable proteins belong to only two distinct protein folds: Ig-like ␤ sandwich fold and ␤-grasp fold.…”

“…A number of proteins of different topologies (6, 10) have been studied for their mechanical properties, and the importance of protein topology to the mechanical stability has emerged (6,(11)(12)(13). It is recognized that the vast majority of mechanically stable proteins identified so far share a common shear topology, in which the two terminal ␤ strands are arranged in parallel and are directly connected to each other by noncovalent interactions (6, 10).…”

“…Upon stretching of these mechanically stable proteins, the two terminal ␤ strands are sheared against each other and the noncovalent interactions connecting the two force-bearing strands provide resistance to mechanical unfolding (6,11,12,(16)(17)(18)(19)(20). These mechanically stable proteins belong to only two distinct protein folds: Ig-like ␤ sandwich fold and ␤-grasp fold.…”