(216d) Single-Molecule Observations of Protein-Protein Interactions At the Solid-Liquid Interface

Protein adsorption at the solid-aqueous interface is a widely studied and very complex phenomenon.    Single-molecule techniques uniquely allow us to separate protein surface dynamics (e.g. protein adsorption, desorption, diffusion) and identify and characterize multiple protein populations and dynamic modes (e.g. oligomeric states, multiple diffusive modes).   Recent work using single-molecule total internal reflection fluorescence microscopy (TIRFM) at low protein concentrations, such that protein-protein interactions are insignificant, has shown protein desorption and diffusion to be highly heterogeneous.  For example, multiple residence time populations were found to correspond to protein oligomeric state where 99.9% of protein monomers reside on the surface for <1s while larger oligomers (trimers and tetramers) reside for up to hundreds of seconds.  These findings suggest that protein-protein interactions, not just protein-surface interactions, must greatly influence protein layer formations and protein surface dynamics observed at high concentrations where protein unfolding and aggregation is thought to occur.  By combining TIRFM and intermolecular resonance energy transfer (RET), the dynamics of protein-protein associations were directly observed and quantified at the solid-liquid interface.   Using this technique, dynamic associations of two or more bovine serum albumin (BSA) molecules were found to be heterogeneous and fast with the majority (80-90%) of protein-protein associations lasting for <1s.  Based on a distribution of RET efficiency (related to the extent of protein association), protein molecules were identified as unassociated, partially associated, or completely associated.  The RET efficiency, or extent of association, are interpreted as clusters (complete RET) and free monomers (no RET).  For increasing bulk protein solution concentrations the fraction of time a molecule on average spent completely associated increased while the fraction of time spent unassociated decreased.  Surprisingly, the distributions of times spent completely associated remained constant for all concentrations.  These observations suggest a pre-layer formation model of heterogeneous protein surface coverage, consisting clusters and free monomers, where local surface dynamics (near clusters or in free area) are independent of surface coverage.