Protein denaturation at the solid-liquid interface is a concern for many fields including biocatalysis, biosensing, biomaterials, and pharmaceuticals. Typically this phenomenon has been studied through ensemble measurements, averaging the heterogeneous behaviors of populations that make up the sample, losing information on population variability. Here we apply single-molecule fluorescence microscopy, protein engineering techniques, and high-throughput image analysis and particle tracking in order to study large numbers (~10<sup>5</sup>) of individual molecules of the model protein T4 lysozyme upon adsorption at the solid-liquid interface. Site-specific labeling of the protein with pairs of fluorophores through site-directed non-natural amino acid incorporation provides simultaneous structural information via Forster Resonance Energy Transfer (FRET). This method of label incorporation allows placement of FRET pairs within different subdomains of T4 lysozyme, providing a method to study the denaturation of specific structural motifs within the model protein. Mutants of T4 lysozyme have been cloned and expressed in order to label non-perturbing locations within the n-terminal catalytic subdomain, the c-terminal substrate binding subdomain, as well as a mutant to study unfolding between subdomains. Utilization of self-assembled monolayers (SAMs) provides a method for tuning the chemistry of the interface, allowing study of a model hydrophobic surface trimethylsilane, the hydrophilic surface fused silica, and polyethylene glycol brushes, a model for protein resistance. Experiments with these surfaces and mutants will probe the effect of surface chemistry on protein denaturation for specific subdomains within lysozyme. Finally, by changing the concentration of bulk lysozyme in solution, the effect of protein-protein interactions at the interface on the rate of unfolding will be determined.

Probing protein denaturation at the solid-liquid interface with single-molecule fluorescent microscopy

249th National Meeting (2015)

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