Single-Molecule Hydrophobic Interactions: From Experiment to Simulation and Equilibrium to Non-Equilibrium

Hydrophobic interactions (HIs) are a fundamental driving force in nature, determining the structure and self-assembly characteristics of many of the molecules of life. Generally, theories and simulations pertaining to hydrophobic solvation and interactions have focused on small length-scale physics, while experimental efforts have observed hydrophobic interactions involving macroscopic systems. Here, we investigate the HI using a highly effective model system: a glycine-serine repeat peptide, functionalized with a varying number of hydrophobic leucine groups, that interacts with a hydrophobic self-assembled monolayer surface. Simulations are performed hand-in-hand with single-molecule atomic force microscopy, constituting a unique convergence of theory and experiment for directly probing the HI. Both sets of results indicate a free energy of peptide removal that scales linearly with the number of leucine residues, at around 3.4 k_BT/leucine for experiment and ranging from 1.6 to 5.6 k_BT/leucine in simulations. The simulated systems closely mirror the experimental set-up, even employing the same non-equilibrium technique, specifically Jarzynski’s equality, to evaluate free energy differences. In addition to agreeing well with experiment, our simulations also identify key factors driving this observed scaling. While all peptides are relatively disordered in solution and absorbed to surfaces, we observe differences in the tetrahedral structure of nearby water, as well as in changes in the solvent accessible surface area of hydrophobic residues. Based on these same metrics, we find signatures of both small- and large-scale hydrophobic solvation theories. Additionally, non-equilibrium simulations provide a detailed dynamical view of how peptides detach from the surface. In accord with the idea that the free energy, and thus the work to remove the peptides, is dominated by hydrophobic association, the greatest contribution to the non-equilibirum work is found to come from the process of detaching leucine residues from the surface.