  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 work 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.
