  Single-molecule force spectroscopy has opened a new field of research in
molecular biophysics and biochemistry. Pulling experiments on individual
proteins permit us to monitor conformational transitions with high temporal
resolution and measure their free energy landscape. The force-extension curves
of single proteins often present large hysteresis, with unfolding forces that
are higher than refolding ones. Therefore, the high energy of the transition
state (TS) in these molecules precludes kinetic rates measurements in
equilibrium hopping experiments. In irreversible pulling experiments,
force-dependent kinetic rates measurements show a systematic discrepancy
between the sum of the folding and unfolding TS distances derived by the
kinetic Bell-Evans model and the full molecular extension predicted by elastic
models. Here, we show that this discrepancy originates from the force-induced
movement of TS. Specifically, we investigate the highly kinetically stable
protein barnase, using pulling experiments and the Bell-Evans model to
characterize the position of its kinetic barrier. Experimental results show
that while the TS stays at a roughly constant distance relative to the native
state, it shifts with force relative to the unfolded state. Interestingly, a
conversion of the protein extension into amino acid units shows that the TS
position follows the Leffler-Hammond postulate: the higher the force, the lower
the number of unzipped amino acids relative to the native state. The results
are compared with the quasi-reversible unfolding-folding of a short DNA
hairpin.
