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Show 77 college of science Protein-protein binding interactions are widely studied because of their key role in regulation of cel-lular processes. Existing methods for measuring protein-protein binding affinities utilize high density ensemble measurements which are hindered by molecular transport to the capture surface. Little work has been done to study the kinetics of bio-molecular interactions at the single molecule level. In collaboration with the Harris group (Department of Chemistry), the Jorgensen group has developed a new method for measuring protein-protein binding affinities called kinetic imaging of single molecule interactions (KISMI). KISMI uses TIRF microscopy to investigate discrete binding events between a pro-tein immobilized covalently on a glass surface and a free protein in solution. Utilizing a low density capture surface, KISMI overcomes transport limitations associated with alternative methods (SPR and microarrays). As a result, KISMI has the ability to accurately detect nanomolar to sub-nanomolar KD's. Specifically, this project has focused on (a) the binding of two SNARE proteins, syntaxin and synapto-brevin, which are involved in synaptic vesicle exocytosis, and (b) IgG antibody-antigen interactions. Because single events are observed, it is essential that the capture surface be resistant to non-specific protein adsorption. In order to investigate non-specific protein-surface interactions, a variety of cova-lently functionalized surfaces were prepared and tested. By screening modified surfaces for non-spe-cific protein adsorption, it was found that in comparison to an unmodified surface, a fluorobenzamide modified surface decreased non-specific SNARE-surface binding by 95% while an acetamide modified surface decreased non-specific IgG antibody adsorption by 99%. By creating discrete protein binding sites within these functionalized surfaces, KISMI technology can be used to accurately measure the kinetics involved in either SNARE complex formation or IgG antibody-antigen binding. INVESTIGATION OF THE KINETICS OF PROTEIN-PROTEIN BINDING USING SINGLE MOLECULE IMAGING Jenna Whippen (Erik Jorgensen) Department of Biology University of Utah UNDERGRADUATE RESEARCH ABSTRACTS Jenna Whippen Erik Jorgensen |