Single-Molecule Investigation of Surface Heterogeneity and Crowding on Protein Conformation at the Solid-Liquid Interface

Protein-surface interactions are a concern for a number of fields, including biocatalysis, biosensing, biomaterials, and chromatography. Typically these interactions have been studied through ensemble measurements, averaging the heterogeneous behaviors of individual molecules. Here we apply single-molecule fluorescence microscopy and protein engineering techniques in order to study large numbers (10⁴-10⁵) of individual T4 lysozyme proteins upon adsorption at the silica-water interface. Site-specific labeling of this model protein with pairs of fluorophores provides simultaneous structural information via Forster Resonance Energy Transfer (FRET). Analysis of individual molecular trajectories with FRET provides quantitative information about protein adsorption, diffusion, and desorption as a function of protein folded state. Super-resolution mapping of the surface reveals nanoscale heterogeneities on the fused silica surface, despite careful sample preparation. At low protein concentration, the diffusive behavior of proteins is dominated by adsorption and immobilization of unfolded protein to a small number of surface sites. As the concentration of protein on the surface increases, these strong adsorption sites become passivated and the surface appears more homogeneous. Further increase in protein concentration increases the rate of protein unfolding, suggesting crowding effects on the surface may be denaturing for T4 lysozyme on fused silica.