Acceleration of Proteolytic Activity Associated with Selection of Thiol Ligand Coatings on Quantum Dots

Nanoparticle bioconjugates are attractive probes for measuring the activity of hydrolytic enzymes. In these configurations, the localization of multiple copies of a hydrolase substrate to a nanoparticle scaffold has been reported to enhance apparent activity by factors of 2 to 3 compared to that for equivalent amounts of substrate in bulk solution. Here, we studied the effect of surface chemistry on protease activity using multivalent QD-peptide substrate conjugates as a model system. QDs were coated with cysteine (CYS), glutathione (GSH), dihydrolipoic acid (DHLA), or 3-mercaptopropionic acid (MPA) ligands, and thrombin and trypsin were used as model proteases. Proteolytic activity was measured for different combinations of ligand and protease using Förster resonance energy transfer (FRET)-based assays. The highest levels of activity were observed with CYS and GSH coatings, and the lowest levels of activity were observed with DHLA and MPA coatings. In all cases, proteolytic activity was accelerated compared to that for an equivalent amount of substrate in bulk solution, with up to 80- and 65-fold increases in the apparent specificity constants for thrombin and trypsin, respectively. Thrombin was more strongly affected by the QD surface chemistry, with up to a 50-fold variation in its apparent specificity constant between ligand coatings, whereas only a 5-fold variation was observed with trypsin. These trends were correlated to adsorption of the proteases on the QDs and are discussed in the context of the physicochemical properties of both components. This work clearly indicates a critical role for the nanoparticle interface in mediating substrate turnover and provides some of the strongest support to date for a so-called hopping model of activity.