Viscoelasticity of a single poly-protein probed step-by-step during its mechanical unfolding and refolding under the force-clamp conditions.

One of still outstanding issues in protein folding is to be able to directly observe structural changes occurring along the folding pathway. Herein, we report on changes of the viscoelastic properties for a single protein molecule measured along its mechanically-induced unfolding and refolding trajectories. We use a model system, the I27 poly-protein, and investigate its conformational changes force-clamp AFM (FC-AFM) spectroscopy. Typically only protein's length and force have been measured using this approach. By applying Euler-Bernoulli model of the AFM cantilever with properly accounted hydrodynamic damping we show how to access - from the same measurements - related changes of two additional observables such as molecular stiffness and molecular friction coefficient. Our results are compared to recent analytical models and experimental results. These findings are expected to lead to proper identification of the intermediate folding states from the knowledge of their mechanical properties.