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nanoparticle-protein conjugates are not easily crystallized. Previous work from our group suggests that the GB3 protein remains globular when adsorbed to gold nanoparticles (AuNPs), but it is unclear whether the tertiary structure is retained. Here, we apply several novel NMR-based approaches to probe the structure and orientation of GB3 bound to AuNPs. We have developed a method for monitoring hydrogen-deuterium exchange (HDX) on the AuNP surface, and we find that HDX rates of surface-bound GB3 are highly correlated with GB3 in solution. Overall, rates are approximately 20 times slower for the adsorbed protein, suggesting that GB3 is stabilized and largely retains its native structure on the surface. Methyl labeling of lysine residues suggests that the orientation of GB3 is fixed on the AuNP, with the helical face exposed to solution. Using differential isotopic labeling, we have determined that adsorbed GB3 molecules do not readily exchange with GB3 in solution, and any exchange that happens occurs on a timescale much longer than 18 hr. These experiments provide strong structural evidence that GB3 adopts a stable, native-like fold and orientation on the AuNP surface, and they open the door for future investigations of protein structure on surfaces.

Measuring volatile phenols in wine is essential in ensuring superior wine quality. A new analytical technique, called solid-phase mesh-enhanced sorption from headspace (SPMESH), was modified with direct immersion (DI) conditions and coupled to direct analysis in real time–mass spectrometry (DART–MS) to be used to detect smoke taint in winemaking.