Estimating the bending modulus of a FtsZ bacterial-division protein filament.

FtsZ, a cytoskeletal protein homologous to tubulin, is the principle constituent of the division ring in bacterial cells. It is known to have force-generating capacity in vitro and has been conjectured to be the source of the constriction force in vivo. Several models have been proposed to explain the generation of force by the Z ring. Here we re-examine data from in vitro experiments in which Z rings formed and constricted inside tubular liposomes, and we carry out image analysis on previously published data with which to better estimate important model parameters that have proven difficult to measure by direct means. We introduce a membrane-energy-based model for the dynamics of multiple Z rings moving and colliding inside a tubular liposome and a fluid model for the drag of a Z ring as it moves through the tube. Using this model, we estimate an effective membrane bending modulus of 500-700 pN nm. If we assume that FtsZ force generation is driven by hydrolysis into a highly curved conformation, we estimate the FtsZ filament bending modulus to be 310-390 pN nm(2). If we assume instead that force is generated by the non-hydrolysis-dependent intermediate curvature conformation, we find that B(f)>1400 pN nm(2). The former value sits at the lower end of the range of previously estimated values and, if correct, may raise challenges for models that rely on filament bending to generate force.

The molecular mechanism underlying mechanical anisotropy of the protein GB1.

Mechanical responses of elastic proteins are crucial for their biological function and nanotechnological use. Loading direction has been identified as one key determinant for the mechanical responses of proteins. However, it is not clear how a change in pulling direction changes the mechanical unfolding mechanism of the protein. Here, we combine protein engineering, single-molecule force spectroscopy, and steered molecular dynamics simulations to systematically investigate the mechanical response of a small globular protein GB1. Force versus extension profiles from both experiments and simulations reveal marked mechanical anisotropy of GB1. Using native contact analysis, we relate the mechanically robust shearing geometry with concurrent rupture of native contacts. This clearly contrasts the sequential rupture observed in simulations for the mechanically labile peeling geometry. Moreover, we identify multiple distinct mechanical unfolding pathways in two loading directions. Implications of such diverse unfolding mechanisms are discussed. Our results may also provide some insights for designing elastomeric proteins with tailored mechanical properties.

Enhancing the mechanical stability of proteins through a cocktail approach.

Rationally enhancing the mechanical stability of proteins remains a challenge in the field of single molecule force spectroscopy. Here we demonstrate that it is feasible to use a "cocktail" approach for combining more than one approach to enhance significantly the mechanical stability of proteins in an additive fashion. As a proof of principle, we show that metal chelation and protein-protein interaction can be combined to enhance the unfolding force of a protein to ∼450 pN, which is >3 times of its original value. This is also higher than the mechanical stability of most of proteins studied so far. We also extend such a cocktail concept to combine two different metal chelation sites to enhance protein mechanical stability. This approach opens new avenues to efficiently regulating the mechanical properties of proteins, and should be applicable to a wide range of elastomeric proteins.