Measuring the energy landscape roughness and the transition state location of biomolecules using single molecule mechanical unfolding experiments

TL;DR

This paper introduces a method to measure the roughness of biomolecular energy landscapes using temperature variation in single-molecule unfolding experiments, enhancing understanding of transition states without assuming a specific reaction coordinate.

Contribution

It presents a formalism that allows direct measurement of energy landscape roughness from experimental data, and discusses challenges in accurately determining transition state locations.

Findings

Roughness of energy landscapes can be measured without assuming reaction coordinates.

Transition state location determination is complex and depends on the molecule's brittleness.

Temperature variation provides insights into energy landscape features in unfolding experiments.

Abstract

Single molecule mechanical unfolding experiments are beginning to provide profiles of the complex energy landscape of biomolecules. In order to obtain reliable estimates of the energy landscape characteristics it is necessary to combine the experimental measurements with sound theoretical models and simulations. Here, we show how by using temperature as a variable in mechanical unfolding of biomolecules in laser optical tweezer or AFM experiments the roughness of the energy landscape can be measured without making any assumptions about the underlying reaction oordinate. The efficacy of the formalism is illustrated by reviewing experimental results that have directly measured roughness in a protein-protein complex. The roughness model can also be used to interpret experiments on forced-unfolding of proteins in which temperature is varied. Estimates of other aspects of the energy landscape such as free energy barriers or the transition state (TS) locations could depend on the precise model used to analyze the experimental data. We illustrate the inherent difficulties in obtaining the transition state location from loading rate or force-dependent unfolding rates. Because the transition state moves as the force or the loading rate is varied it is in general difficult to invert the experimental data unless the curvature at the top of the one dimensional free energy profile is large, i.e the barrier is sharp. The independence of the TS location on force holds good only for brittle or hard biomolecules whereas the TS location changes considerably if the molecule is soft or plastic. We also comment on the usefulness of extension of the molecule as a surrogate reaction coordinate especially in the context of force-quench refolding of proteins and RNA.