Use of angle-resolved surface plasmon resonance imaging (SPRi) for the characterization of protein binding dynamics

Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Protein-protein interactions are essential to multiple cellular functions. However, the individual mechanisms that control protein-protein interactions are not clearly understood, and the transient dynamics of multi-protein complexes are challenging to study. The work presented in this thesis focuses on improvements to angle-resolved surface plasmon resonance imaging (SPRi) methods, including surface fabrication and patterning techniques, which enable multi-array kinetic and thermodynamic studies of protein binding events on surfaces. Because immobilization can significantly influence binding mechanisms, we investigated the effects of density and orientation of surface attached proteins on binding efficiency and kinetics. The activity of trimeric cytokine Tumor Necrosis Factor Alpha (TNFa), a model system, was highly dependent on immobilization conditions. Using a unique multi-wavelength SPRi approach to simultaneously determine dielectric constants and thicknesses of TNFa layers we were able to distinguish between different oligomeric states of a pre-associated multi-protein complex immobilized on the surface. For a different protein-protein system, IKKB binding to NF-kB Essential Modulator (NEMO), we generated a library of IKKB mutants and determined activity using both solution and surface assays. Results for IKKB mutants patterned on the biosensor surface agreed well with solution-phase fluorescence anisotropy measurements. We quantified the contribution of select IKKB residues on the specificity of NEMO binding and identified two new hot spot regions. These results may aid the development of inhibitors for IKKB:NEMO binding in targeted drug discovery. The experimental capabilities used for the above protein studies were optimized on an unrelated non-biological system. For that case, we developed a general methodology to characterize the wavelength dependent optical properties of noble-metal nanoparticles (NPs) in close proximity to a metal sensor surface. NPs are often used as labels to enhance the sensitivity of SPR measurements, however, the dependence of NP optical properties on the distance of the NP to a metal substrate is not fully understood. We report the optical properties of 10 nm gold NPs as a function of particle-to-metal substrate distance and excitation wavelength. These results may aid predictive theoretical models of the signal-enhancing capabilities of NPs. This new knowledge could lead to the development of higher sensitivity SPR biosensors.2031-01-0