Single-molecule studies in which a mechanical force is transmitted to the molecule of interest and the molecular extension or position is monitored as a function of time are versatile tools for probing the dynamics of protein folding, stepping of molecular motors, and other biomolecular processes involving activated barrier crossing. One complication in interpreting such studies, however, is the fact that the typical size of a force probe (e.g., a dielectric bead in optical tweezers or the atomic force microscope tip/cantilever assembly) is much larger than the molecule itself, and so the observed molecular motion is affected by the hydrodynamic drag on the probe. This presents the experimenter with a nontrivial task of deconvolving the intrinsic molecular parameters, such as the intrinsic free energy barrier and the effective diffusion coefficient exhibited while crossing the barrier from the experimental signal. Here we focus on the dynamical aspect of this task and show how the intrinsic diffusion coefficient along the molecular reaction coordinate can be inferred from single-molecule measurements of the rates of biomolecular folding and unfolding. We show that the feasibility of accomplishing this task is strongly dependent on the relationship between the intrinsic molecular elasticity and that of the linker connecting the molecule to the force probe and identify the optimal range of instrumental parameters allowing determination of instrument-free molecular dynamics.
Keywords: Kramers theory; protein folding; single-molecule force-probe spectroscopy.
© 2015 The Protein Society.