  We analyze free energy estimators from simulation trials mimicking
single-molecule pulling experiments on a neck linker of a kinesin motor. For
that purpose, we have performed a version of steered molecular dynamics (SMD)
calculations. The sample trajectories have been analyzed to derive distribution
of work done on the system. In order to induce unfolding of the linker, we have
stretched the molecule at a constant pulling force and allowed for a subsequent
relaxation of its structure. The use of fluctuation relations (FR) relevant to
non-equilibrium systems subject to thermal fluctuations allows us to assess the
difference in free energy between stretched and relaxed conformations. To
further understand effects of potential mutations on elastic properties of the
linker, we have performed similar in silico studies on a structure formed of a
polyalanine sequence (Ala-only) and on three other structures, created by
substituting selected types of amino acid residues in the linker's sequence
with alanine (Ala) ones. The results of SMD simulations indicate a crucial role
played by the Asparagine (Asn) and Lysine (Lys) residues in controlling
stretching and relaxation properties of the linker domain of the motor.
