Residue-Specific Dock-Loosen-Unfold Mechanism of GB1 on Nanoparticle Surfaces Revealed by Kinetic and Φ-Value Analysis

Nanoparticles interact dynamically with proteins, often leading to adsorption-induced conformational changes that alter protein function and contribute to corona formation. Here we investigated the adsorption and unfolding of a model protein GB1 on latex nanoparticle surfaces using a combination of mutational analysis, equilibrium binding assays, stopped-flow kinetics and Φ-value interpretation. Seven site-directed variants of GB1 were studied to dissect residue-specific contributions to adsorption energetics. Fluorescence binding isotherms revealed that D46A and T53A mutations weakened surface affinity, while kinetic analysis demonstrated that D46A reduced adsorption rate by ~6-fold and produced a dramatic unfolding/refolding shift, identifying Asp46 as a key docking site. Φ-value analysis further highlighted Asp46 and Thr53 as central residues in the adsorption transition state, whereas mutations in the hydrophobic core or distal loops had negligible effects. These results support a dock-loosen-unfold mechanism in which electrostatic recognition initiates binding, followed by hydrophobic exposure and hairpin stabilization. This residue-level sampling of key sites advances mechanistic understanding of protein-nanoparticle interactions and suggests strategies for tuning surface charge to control corona formation. Our approach provides a generalizable method to map adsorption transition states, with implications for designing safer nanomaterials, predicting protein corona composition, and harnessing protein unfolding in biosensing applications.