Improved Free-Energy Landscape Quantification Illustrated with a Computationally Designed Protein-Ligand Interaction

Abstract

Quantifying the energy landscape underlying protein-ligand interactions leads to an enhanced understanding of molecular recognition. A powerful yet accessible single-molecule technique is atomic force microscopy (AFM)-based force spectroscopy, which generally yields the zero-force dissociation rate constant (k_off ) and the distance to the transition state (Δx^≠ ). Here, we introduce an enhanced AFM assay and apply it to probe the computationally designed protein DIG10.3 binding to its target ligand, digoxigenin. Enhanced data quality enabled an analysis that yielded the height of the transition state (ΔG^≠ =6.3±0.2 kcal mol^-1 ) and the shape of the energy barrier at the transition state (linear-cubic) in addition to the traditional parameters [k_off (=4±0.1×10^-4 s^-1 ) and Δx^≠ (=8.3±0.1 Å)]. We expect this automated and relatively rapid assay to provide a more complete energy landscape description of protein-ligand interactions and, more broadly, the diverse systems studied by AFM-based force spectroscopy.

Keywords: atomic force microscopy; energy landscape; protein design; protein-ligand interactions; single-molecule force spectroscopy.