(720a) Surface Chemistry Effects on Dynamic Interfacial Protein-Protein Association

Surface chemistry modification has been exploited in many applications including bioseparations, biomaterials, and biosensing in order to tune protein adsorption, layer formation, and aggregation.  In particular, polyethylene glycol (PEG) surfaces are commonly acknowledged to reduce protein adsorption and layer formation when compared with hydrophobic surfaces.  However there is still much debate over the mechanism of PEG’s ‘protein resistance’ and more generally about how surface chemistry affects protein interfacial dynamics.  Unlike ensemble-averaging methods, single-molecule (SM) methods capture spatial and population heterogeneity (monomers vs. oligomers) as well as separate specific dynamics (measure adsorption and desorption independently).  In previous work, we used SM total internal reflection fluorescence microscopy (TIRFM) to study isolated protein-surface interactions of fibrinogen (Fg) with PEG and hydrophobic trimethyl silane (TMS) surfaces.  Fg adsorption and desorption were found to be highly dynamic (desorbing in seconds) and heterogeneous (dependent on oligomer size).  Unexpectedly, Fg molecules desorbed more slowly from PEG surface than from TMS surfaces suggesting that isolated protein-surface interactions alone cannot explain PEG’s ‘protein resistance’.  Based on these observations, we hypothesized that surface chemistry plays an important role in influencing interfacial protein-protein interactions.

In our current work, we combined SM-TIRFM and intermolecular resonance energy transfer (RET) to directly observe the dynamics of Fg self-association on both oligoethylene glycol (OEG) and hydrophobic TMS surfaces.  On both surfaces we found evidence of dynamic Fg self-associations. These interfacial associations were reversible and fast (the vast majority of associations lasted for less than 5s).  Protein self-associations at the interface also resulted in longer surface residence times, leading to more opportunities for protein oligomerization and aggregation at the interface.  These findings were in stark contrast to previously proposed protein interfacial nucleation and growth models of irreversible adsorption and slow aggregation in which surface chemistry appeared mainly to influence adsorption.  Instead, SM-TIRFM and intermolecular RET findings suggested that interfacial Fg aggregates form via a dynamic nucleation and (reversible) growth process where fast dynamic processes, influenced by surface chemistry, are subtly balanced with rare aggregation events.