Experimental free energy surface reconstruction from single-molecule force spectroscopy using Jarzynski's equality.

We used the atomic force microscope to manipulate and unfold individual molecules of the titin I27 domain and reconstructed its free energy surface using Jarzynski's equality. The free energy surface for both stretching and unfolding was reconstructed using an exact formula that relates the nonequilibrium work fluctuations to the molecular free energy. In addition, the unfolding free energy barrier, i.e., the activation energy, was directly obtained from experimental data for the first time. This Letter demonstrates that Jarzynski's equality can be used to analyze nonequilibrium single-molecule experiments, and to obtain the free energy surfaces for molecular systems, including interactions for which only nonequilibrium work can be measured.