Single-Molecule Force Spectroscopy of Multimodular Proteins: A New Method to Extract Kinetic Unfolding Parameters

Abstract

When multimodular proteins composed of a number of identical modules are unfolded in the course of a single-molecule force-spectroscopy experiment, both the probability distribution of the unfolding force and the most probable unfolding force depend on the number of not-yet-unfolded modules in the protein. We show that this unfolding can be described in the framework of a generalized Bell-Evans model, based on certain minimal assumptions concerning the force loading conditions. In particular, our modified equations for the unfolding force predict, for a multimodular protein containing N not-yet-unfolded modules, a linear dependence of the most probable unfolding force on ln(N). This conclusion has been confirmed in experiments with GB1 proteins, which verify in addition further consequences of the proposed model, including a modified probability distribution of the unfolding force. Based on these results, we propose a new method for analyzing single-molecule force-spectroscopy data for multimodular proteins, which enables one to extract kinetic unfolding parameters from experimental data with no additional assumptions about the unfolding process.