A Computationally Designed Protein-Ligand Interaction is Mechanically Robust

Abstract

Quantifying the energetics and dynamics protein-ligand interactions is critical to understanding molecular recognition. Computational design provides an exciting means by which to expand the repertoire of naturally existing protein-ligand interactions for diverse biomedical applications. In both cases, rapid and precise determination of the fundamental physical parameters that govern the strength of a protein-ligand bond is needed. A particularly powerful technique is single molecule force spectroscopy (SMFS), which generally reports the off-rate at zero applied force (koff) and the distance to the transition state (Δx‡). Here, we used constant-force SMFS to characterize the computationally designed protein DIG10.3 binding to its target ligand, the steroid digoxigenin. To improve the precision and throughput of molecular recognition studies using atomic force microscopy, we integrated a number of technical improvements, including low-drift cantilevers, site-specific coupling, and corrected pulling geometries. Our enhanced precision allowed us to not only determine koff (= 4 ± 0.1×10−4 s−1) and Δx‡ (= 8.3 ± 0.1 A,), but also the height of the transition state (ΔG‡ = 6.3 ± 0.2 kCal/mol) and the shape of the energy barrier at the transition sate (linear-cubic potential). Importantly, by doing so in an automated and relatively rapid manner (<2 days of instrument time), we anticipate that these measured energy landscape parameters can provide valuable experimentally feedback to further optimize computational design of protein-ligand interactions.