Paper BI+TF+MI+NS+NC-ThA4
Use of Angle-Resolved SPRi for the Characterization of Protein Binding and Agglomeration Dynamics

Thursday, October 23, 2008, 3:00 pm, Room 202

Transient protein-protein interactions are essential on almost every level of cellular function. In addition, protein aggregates play various roles in cell signaling pathways and have been implicated in the onset of many neurodegenerative conditions such as Alzheimer’s disease. Although various structures of protein complexes have been widely studied, the mechanisms involved in protein binding events are not clearly understood, and the transient dynamics of this formation have proven difficult to study. Investigation of the mechanisms of protein agglomeration and binding, however, is essential to elucidating the role of these structures in diseases. Here we exploit the multi-array quantitative capabilities of angle-resolved surface plasmon resonance imaging (SPRi) to perform kinetic and thermodynamic measurements of protein-protein interactions. Specifically, the agglomeration and small molecule inhibition of Tumor Necrosis Factor (TNF) family members whose primary role is the regulation of immune cells is under investigation. Surface fabrication techniques coupled with multi-channel microfluidic delivery will be employed in order to introduce protein binding partners and small molecules to the surface. Effects of density, orientation, and heterogeneity of surface immobilized protein molecules on protein binding efficiency and kinetics will be investigated and optimum surface fabrication conditions will be identified. In addition, a unique multi-wavelength SPRi approach will be implemented to simultaneously determine dielectric constants and thicknesses of protein layers on a surface. These studies will therefore allow aggregate and nonaggregate structures at the surface to be clearly differentiated. The results of these fundamental studies will allow a broader understanding of how proteins act cooperatively.