While sometimes considered as independent phenomena, interfacial and solution-phase behavior are intrinsically linked by the dynamic exchange of proteins between solution and interfaces.  Protein adsorption and subsequent conformational changes, and/or aggregation at the solid-liquid interface have been suggested to accelerate solution aggregation and contribute to aggregates in solution. By combining single molecule total internal reflection fluorescence microscopy (SM-TIRFM) and intermolecular resonance energy transfer (RET), the dynamics of protein self-associations can be directly observed and quantified at the solid-liquid interface.  Unlike ensemble-averaging methods, single molecule methods uniquely capture both spatial and population heterogeneity (e.g. monomers vs. oligomers).  Using this technique, we examined BSA self-association on poly(ethylene glycol) modified surfaces at low solution concentrations.  We identified three distinct surface states � unassociated monomers, associated dimers, and larger clusters � and directly observed exchanges between these states.   The majority of associations were found to be reversible and fast with rare, but measureable, associations (between monomeric proteins and clusters) remaining associated for much longer times.  While the majority of monomeric proteins desorbed quickly from the surface, some associated proteins resided on the surface for longer time intervals, leading to more opportunities for protein-cluster association and aggregation.  These findings were in stark contrast to previously proposed protein interfacial nucleation and growth models of irreversible adsorption and slow aggregation.  Instead, SM-TIRFM and intermolecular RET findings suggested that interfacial BSA aggregates form via a dynamic nucleation and (reversible) growth process consisting of a subtle balance between fast dynamics and rare aggregation events.

Single-molecule observations of dynamic protein-protein associations at the solid-liquid interface

247th National Meeting (2014)

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