Figure 3.

(A) The use of 1D half filter experiment to quantify binding of two proteins in same solution with AuNPs. GB3 (¹³C labeled) and ubiquitin (Ubq, ¹⁵N labeled) are in the same solution, and 80 nM AuNPs are added. The 15N and 13C filter can be applied to differentiate proton signals originating from each protein independently, and quantitative NMR approaches can be used to measure binding. The residual water signal has been removed for clarity. (B) Backbone amide ¹H-¹⁵N heteronuclear single-quantum coherence (HSQC) experiment to quantify two proteins isotopically labeled the same in the nanoparticle solution. ¹⁵N labeled GB3 and ¹⁵N labeled Ubq are mixed in the same solution with 80 nM AuNP. The peak intensity perturbation measured for 25 μM GB3 and 25 μM Ubq in the presence of AuNP (I, red spectra) compared to the absence of AuNP (I_o, blue spectra). (C) The ¹H-¹⁵N HSQC experiment can be used to determine the binding capacity for each protein by plotting the bound concentration versus various AuNP concentrations. The HSQC spectral intensities are used to determine the bound concentration relative to an external standard. Here, 20 μM GB3 is mixed with 0, 20, 40, 60, and 80 nM AuNP to identify how much is bound to the NP surface. Observed concentrations for GB3 (blue circles) are plotted against the expected values for a folded monolayer of protein on the NP surface (black circles and solid line).