An Experimental Test of Jarzynski's Equality on Free Energy of Protein Unfolding

We performed an experimental test of Jarzynski's equality on the mechanical unfolding of a giant protein, human cardiac titin. Jarzynski's estimator relates the free energy difference ($\Delta G$) between two equilibrium states and the work performed in switching between those states. We used atomic force microscopy to obtain the single-molecule dynamic force spectroscopy of polyproteins consisting of identical tandem repeats of the I27 domain of titin. We found that Jarzynski's equality results in a good estimate of protein unfolding free energy from non-equilibrium measurements.