AFM-Based Force Spectroscopy Provides Quantitative Characterization of Single Residue Contributions in Peptide-Lipid Interactions

Peptide interactions with phospholipid bilayers are vitally important in numerous cellular functions and processes. The challenge in studying these interactions resides in not only the complexity of the biochemical system in question, but the length and time scales in which these interactions take place. When studying these interactions via bulk biochemical assays, one can extract salient information about the peptide(s) being studied, such as the bilayer to solution free energy of transfer. However, mechanical details about the interactions are masked by asynchronous activity. Atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS) has proven to be a robust technique for studying both kinetics and energetics of membrane proteins. Through repeated approach-retraction experiments using an AFM tip functionalized with a peptide, one can probe the binding forces the peptide experiences in proximity to a lipid bilayer. We investigated the interactions between a supported POPC bilayer and two groups of peptides: polyleucines Trp-(Leu)_N, where N=1 to 5 leucines, and pentapeptides Trp-Leu-Leu-Leu-X, where X is a variable guest residue. For each unique peptide, we obtained a rupture force distribution P(F) across all experiments. Analysis of the distributions for each peptide highlights how the rupture force profile changes as a result of increasing (i) peptide length and (ii) hydrophobic content. Complimentary data from coarse-grained molecular dynamics (CG-MD) simulations aid theoretical fitting of P(F), allowing us to extract energetics of the system. Fitting P(F) also demonstrates that forced peptide-lipid dissociation can occur along multiple pathways. Together, this data demonstrates a quantitative approach to analyzing the contributions of single amino acid residues in certain peptide-lipid interactions.