273956 Interfacial Protein Dynamics Resolved Using Single-Molecule Tracking

Wednesday, October 31, 2012: 9:15 AM
413 (Convention Center )
Mark J. Kastantin1, Blake B. Langdon2 and Daniel K. Schwartz2, (1)Chemical and Biological Engineering, University of Colorado, Boulder, CO, (2)Chemical and Biological Engineering, University of Colorado at Boulder, Boulder, CO

Protein adsorption at the solid-liquid interface is fundamental to many applications including biocompatible materials, biofilm fouling, biosensing, and protein separations. Despite abundant research into this topic, a complete mechanistic understanding of dynamic surface behaviors is lacking. The potential for multiple soft (i.e. noncovalent) interactions between protein and surface (e.g. hydrophobic, hydrogen bonding, ionic interactions) often leads to composite behaviors of the average population in which it is difficult to identify the fundamental contributing factors. In contrast to methods that measure average properties (e.g. net adsorption or mean diffusion coefficient), single-molecule methods allow separation of different modes of protein-surface interaction for separate analysis. In this work, total internal reflection fluorescence microscopy is used to attain single-molecule resolution of protein adsorption onto model hydrophilic and hydrophobic solid surfaces using the proteins bovine serum albumin and fibrinogen. Our results demonstrate that heterogeneity is a common theme for all proteins studied on all surfaces.

Proteins were found to adsorb in different aggregation states (i.e. monomers, dimers, etc.) as evidenced by fluorescence intensities that were roughly integer multiples of the lowest observed intensity. Aggregation number was found to subsequently affect their surface affinity and mobility, with larger aggregates exhibiting longer surface residence times and slower diffusion. All protein species were found to be capable of multiple modes of surface interaction as evidenced by multiple observed characteristic residence times and diffusion coefficients, even for monomers. Temperature studies of desorption and diffusion allowed us to determine the apparent Arrhenius activation energy of each mode of surface interaction.

Notably, activation energies of all modes of desorption and diffusion were relatively weak (< 5 kBT). Coupled with the observation that the mean residence time of a single protein is relatively short (~1s) for all proteins on all surfaces, it is apparent that direct protein-surface interactions are insufficient to explain the formation of stable protein layers. Future work will focus on protein-protein interactions and the indirect role of the surface in mediating these interactions.

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See more of this Session: Biomolecules at Interfaces
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