Extension-Dependent Drift Velocity and Diffusion (DrDiff) Directly Reconstructs the Folding Free Energy Landscape of Atomic Force Microscopy Experiments.

Two equilibrium force microscopy trajectories [ q ( t )] of high-precision single-molecule spectroscopy assays were analyzed: the pulling of an HIV RNA hairpin and of a 3-aa sequence of the bacteriorhodopsin membrane protein. Both present hundreds of two-state folding transitions, and their free-energy [ F ( q )] landscapes were previously obtained by deconvolving time signals with the inverse Boltzmann and p _fold methods. In this letter, the two F profiles were reconstructed directly from the measured time-series by the drift-diffusion (DrDiff) framework that characterized the effective conformational drift-velocity [ v ( q )] and diffusion [ D ( q )] coefficients. The two thermodynamic F profiles reconstructed with DrDiff directly from q ( t ) were in good agreement with those previously obtained from the deconvolved time signals. q ( t ) trajectories simulated with a two-dimensional framework in which the diffusion coefficient of the pulling setup ( q coordinate) differed from the molecule ( x coordinate) were also analyzed by DrDiff. The performance in reconstructing F was investigated in different conditions of diffusion anisotropy in the simulated time-series using Brownian dynamics. In addition, recently developed theories were used in order to evaluate the quality of the analysis performed in the experimental time series: the memory effects and the intrinsic biomolecular dynamic properties after connecting the probe to the molecule. With the 2-dimensional diffusive models and the additional analyses, it is proposed that the different physical regimes imposed by the stiffer probes of the two biomolecules will have an impact in the measured extension-dependent D and, thus, in the reconstruction of F by DrDiff. Stiffer AFM probes may reflect the molecular behavior more faithfully and reconstruction of F might be more successful. The reported quantities extracted directly from q ( t ) highlights the current state of the biomolecule characterization by force spectroscopy experiments: it is still challenging despite the recent advances, yet it is very promising.