Protein dimerization probed with site-specific attached single nanoparticles

Self-association of proteins forming dimers or oligomers is a common occurrence in biological systems. Probing the dimerization status can be performed by various size- and mass-selective methods (e.g. analytical ultracentrifugation, gel filtration, ċċċ). Every method has its strengths but generally the detection directly in the solution by optical means is preferred. Metal nanoparticles (NP) can be observed in a light microscope and their surface can be functionalized with different chemical compounds, like dyes or affinity tags. For example, nickel-chelated N-nitrilo-triacetic acid (Ni-NTA) functionalized metal nanoparticles (Ni-NTA NP) can be site-specifically attached to His-tagged proteins, forming a thin single protein layer on a NP. Additional functionalization of the Ni-NTA NP with Raman or fluorescent dyes provide furthermore the ability to observe red-shifted emissions, like surface-enhanced Raman scattering (SERS) or surface-enhanced fluorescence (SEF), especially if plasmonic metals are used as NP material. Those effects are large if single NPs are near to each other upon laser irradiation. In case the NP surface attached proteins form stable dimers or oligomers, the functionalized NPs will be concomitantly linked and in close proximity to each other, forming NP dimers or oligomers itself. The new formed NP-complexes can be detected by dynamic light scattering (DLS), asymmetric flow field fractionation (AFFF) or observed optically on the single-particle level by red-shifted emissions upon laser irradiation. Observing the red-shifted emissions, inelastic scattering (Raman/Mie) or fluorescence, concomitantly with elastic (Rayleigh) emissions via two separate imaging channels in a light-sheet illumination approach allows the check of the NP-dimerization/oligomerization status. This indicates protein interaction in the native suspension environment and enables determination of the ratio of non-oligomerized to oligomerized protein in a microscope image or video. This approach paves the way for fast optical determination of protein-protein or protein-ligand interactions.