(206b) Protein Engineering-Enabled Single Molecule Resolution of Protein Structure At Biomaterial Interfaces

A method was developed to monitor dynamic changes in protein structure and interfacial behavior on surfaces by combining single-molecule (SM)-resonance energy transfer (RET) and protein engineering. This method entails the incorporation of unnatural amino acids to site-specifically label proteins with SM-RET probes for high-throughput dynamic fluorescence tracking microscopy on surfaces. Structural changes in the model protein organophosphorus hydrolase (OPH) were monitored upon adsorption on fused silica (FS) surfaces on a molecule-by-molecule basis. Analysis of >30,000 individual trajectories enabled heterogeneities in the kinetics of surface-induce OPH unfolding to be observed with unprecedented molecular detail. In particular, two distinct pathways were observed; a majority population (~85%) unfolded with a characteristic time scale of 0.10 s, and the remainder unfolded more slowly with a time scale of 0.70 s. Importantly, even following unfolding, OPH readily desorbed from FS surfaces, challenging the generally accepted notion that surface-induced unfolding leads to irreversible protein binding. This suggests that protein fouling of surfaces is a highly dynamic process due to subtle differences in the adsorption/desorption rates of folded and unfolded species. Moreover, such observations imply that surfaces may act as a source of unfolded (i.e. aggregation-prone) protein back into solution. Continuing study of other proteins and surfaces will examine if these conclusions are general or specific to OPH in contact with FS. Ultimately, this method, which is widely-applicable to virtually any protein, provides the framework to develop surfaces and surface modifications with improved biocompatibility.