New method for deciphering free energy landscape of three-state proteins

TL;DR

This paper introduces a novel simulation approach to analyze the free energy landscape of three-state proteins under force, enabling estimation of transition state distances and unfolding barriers with experimental agreement.

Contribution

The study presents a new method to determine transition state positions and barriers in three-state protein unfolding using force-extension simulations and kinetic theory.

Findings

Successfully applied to titin I27 and DDFLN4 proteins

Estimated transition state distances consistent with experimental data

Provided insights into unfolding barriers and intermediate states

Abstract

We have developed a new simulation method to estimate the distance between the native state and the first transition state, and the distance between the intermediate state and the second transition state of a protein which mechanically unfolds via intermediates. Assuming that the end-to-end extension $\Delta R$ is a good reaction coordinate to describe the free energy landscape of proteins subjected to an external force, we define the midpoint extension $\Delta R^*$ between two transition states from either constant-force or constant loading rate pulling simulations. In the former case, $\Delta R^*$ is defined as a middle point between two plateaus in the time-dependent curve of $\Delta R$, while, in the latter one, it is a middle point between two peaks in the force-extension curve. Having determined $\Delta R^*$, one can compute times needed to cross two transition state barriers starting from the native state. With the help of the Bell and microscopic kinetic theory, force dependencies of these unfolding times can be used to locate the intermediate state and to extract unfolding barriers. We have applied our method to the titin domain I27 and the fourth domain of {\em Dictyostelium discoideum} filamin (DDFLN4), and obtained reasonable agreement with experiments, using the C$_{\alpha}$-Go model.