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Use of Angle‐Resolved Surface Plasmon Resonance Imaging (SPRi) for the Characterization of Protein Binding Dynamics

Protein‐protein interactions are essential at almost every level of cellular function. Although structures of various protein complexes have been characterized, the mechanisms involved in protein binding events and the transient dynamics of complex formation are not fully understood. Here we present an improved angle‐resolved surface plasmon resonance imaging (SPRi) methodology that allows real time multi‐array kinetic and thermodynamic analysis of protein binding events on surfaces. Using Tumor Necrosis Factor alpha (TNFα), a homotrimeric protein, as a test system we have developed surface fabrication techniques including multi‐channel microfluidic delivery systems and patterning capabilities, and have employed various surface immobilization strategies to investigate the influences of density, orientation, and heterogeneity of surface immobilized TNFα on protein binding efficiency and kinetics. A unique multi‐wavelength SPRi approach is utilized to simultaneously determine dielectric constants and thicknesses of TNFα layers, allowing characterization of the oligomeric structure of the immobilized trimer. Results showed that TNFα activity for known binding partner TNFR2, as well as TNFα subunit dissociation dynamics, were highly dependent on the selected immobilization conditions. This work was supported by NIGMS and Boston University, Department of Chemistry.