Is End-to-End Distance a Good Reaction Coordinate?

Abstract

Single-molecule force spectroscopy opens a new door for investigating detailed biomolecular interactions and their thermodynamic properties by pulling molecules apart while monitoring the force exerted on them. Recent advances in the nonequilibrium work theorem allows one to determine the free energy landscapes of these processes. However, the resulting free energy surfaces are expressed as a function of molecular end-to-end distance, which differs from the normal reaction coordinate, and raises the question whether molecular extension is a good order parameter. Using unfolding of the I27 domain of human cardiac titin, we show that the molecular end-to-end distance is a good reaction coordinate by comparing the free energies determined from force spectroscopy to traditional bulk chemical studies. The trends in protein unfolding free energies are consistent for force spectroscopy and bulk chemical studies, where the reaction coordinate is well-defined. The results demonstrate that the information from single-molecule pulling experiments are meaningful and useful for understanding the mechanism of protein folding.