The active sites of subtilisin and trypsin have been studied by paired IR spectroscopic and X‐ray crystallographic studies. The active site serines of the proteases were reacted with 4‐cyanobenzenesulfonyl fluoride (CBSF), an inhibitor that contains a nitrile vibrational reporter. The nitrile stretch vibration of the water‐soluble inhibitor model, potassium 4‐cyanobenzenesulfonate (KCBSO), and the inhibitor were calibrated by IR solvent studies in H2O/DMSO and the frequency‐temperature line‐slope (FTLS) method in H2O and THF. The inhibitor complexes were examined by FTLS and the slopes of the best fit lines for subtilisin‐CBS and trypsin‐CBS in aqueous buffer were both measured to be −3.5x10−2 cm−1/°C. These slopes were intermediate in value between that of KCBSO in aqueous buffer and CBSF in THF, which suggests that the active‐site nitriles in both proteases are mostly solvated. The X‐ray crystal structures of the subtilisin‐CBS and trypsin‐CBS complexes were solved at 1.27 and 1.32 Å, respectively. The inhibitor was modelled in two conformations in subtilisin‐CBS and in one conformation in the trypsin‐CBS. The crystallographic data support the FTLS data that the active‐site nitrile groups are mostly solvated and participate in hydrogen bonds with water molecules. The combination of IR spectroscopy utilizing vibrational reporters paired with X‐ray crystallography provides a powerful approach to studying protein structure.

reaction boundaries of successively lower velocity and number of components. We report algebraic relationships for velocities and composition. The concentration space is subdivided into regions exhibiting different boundary patterns with discontinuous transitions along characteristic subspaces. The extension of the effective particle theory provides physical insights into the coupled co-migration processes, and can be used to interpret boundary patterns derived from experimentally measured sedimentation coefficient distributions. It can be used to plan multi-component sedimentation velocity experiments to determine binding constants and complex stoichiometries.

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.

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