Polyprotein of GB1 is an ideal artificial elastomeric protein

Abstract

Naturally occurring elastomeric proteins function as molecular springs in their biological settings and show mechanical properties that underlie the elasticity of natural adhesives, cell adhesion proteins and muscle proteins. Constantly subject to repeated stretching-relaxation cycles, many elastomeric proteins demonstrate remarkable consistency and reliability in their mechanical performance. Such properties had hitherto been observed only in naturally evolved elastomeric proteins. Here we use single-molecule atomic force microscopy techniques to demonstrate that an artificial polyprotein made of tandem repeats of non-mechanical protein GB1 has mechanical properties that are comparable or superior to those of known elastomeric proteins. In addition to its mechanical stability, we show that GB1 polyprotein shows a unique combination of mechanical features, including the fastest folding kinetics measured so far for a tethered protein, high folding fidelity, low mechanical fatigue during repeated stretching-relaxation cycles and ability to fold against residual forces. These fine features make GB1 polyprotein an ideal artificial protein-based molecular spring that could function in a challenging working environment requiring repeated stretching-relaxation. This study represents a key step towards engineering artificial molecular springs with tailored nanomechanical properties for bottom-up construction of new devices and materials.

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

“…The number of domains in a polyprotein affects the measured mechanical stability, but this effect is small (36,37). Hence, our measured mechanical stability of Top7 using a heteropolyprotein does not differ from the value expected from a polyprotein (Top7) 8 . SMD Simulations.…”

“…Our designed polyprotein chimera allows us to directly compare the mechanical stability of Top7 with that of GB1 (8,12). For the force-extension curves of the polyprotein chimera, we observed that the mechanical unfolding events of Top7 scattered among the GB1 unfolding events: the mechanical unfolding of Top7 can occur before any GB1 unfolding event or quite often after some of the GB1 domains have been unfolded.…”

“…2A). In this polyprotein chimera, the well characterized protein GB1 (8,12), whose mechanical unfolding is characterized by a contour length increment ⌬L C of Ϸ18 nm and an unfolding force of Ϸ180 pN (at pulling speed of 400 nm/s), serves as an internal marker for identifying single-molecule stretching events as well as pinning down the signature of the mechanical unfolding of Top7.…”

“…Recent development in single-molecule force spectroscopy has made it possible to study the mechanical properties of proteins at singlemolecule level (5,6). These studies not only revealed rich information about the architectural design of elastomeric proteins and shed light on the biophysical principles underpinning various biological processes, but also revealed promising prospect of using engineered elastomeric proteins for nanomechanical applications (7)(8)(9). However, in comparison to chemical and thermodynamic properties of proteins, experimental data on mechanical properties of proteins remains rather limited and molecular determinants of mechanical properties of proteins are less well understood.…”

Biomimetic materials for tissue engineering

“…ELPs are promising scaffolding materials for certain tissue engineering applications [34]. Inspired by the natural elastomeric proteins, a small nonmechanical protein GB1 (the streptococcal B1 immunoglobulin-binding domain of protein G) was recently engineered into a polyprotein (GB1) 8 and was found to have excellent elastomeric properties [35], which could be a candidate material for tissue engineering scaffolds.…”