Optimization and Changes in the Mode of Proteolytic Turnover of Quantum Dot–Peptide Substrate Conjugates through Moderation of Interfacial Adsorption

Abstract

Enzymes have many important roles in biology and industry, and proteases are one of the most important classes of enzymes. Semiconductor quantum dots (QDs) are attractive materials for developing protease activity probes because of their advantageous physical and optical properties; however, interactions between a protease and a QD conjugated with its substrate can affect the turnover of that substrate. Here, we study the turnover of multivalent QD-peptide substrate conjugates as a function of multiple parameters: (i) the ligand coating on the QD, including dihydrolipoic acid (DHLA), glutathione (GSH), DHLA-poly(ethylene glycol) (DHLA-PEG), and DHLA-zwitterionic sulfobetaine (DHLA-SB); (ii) the identity of the protease, including trypsin, thrombin, and plasmin; and (iii) the number of substrate and nonsubstrate biomacromolecules conjugated per QD. We show that limiting protease adsorption on QDs is critical for optimizing the turnover of conjugated peptide substrates. Protease adsorption is inhibitory, and very strong adsorption leads to an apparent "scooting" mode of activity with limited turnover. In contrast, with weaker adsorption, enhancements in the turnover rate likely result from a "hopping" mode of activity. The putative hopping mode is thought to feature processive turnover of all substrates in multivalent conjugates with a rate-limiting step of diffusion between individual conjugates, and the magnitude of such enhancements increases with decreases in adsorption. Although it was possible to passivate DHLA- and GSH-coated QDs with high densities of conjugated biomacromolecules, the most effective strategy for reducing adsorption was the substitution of these ligands. Whereas passivation incrementally increased turnover, DHLA-PEG and DHLA-SB ligands converted the mode of turnover with plasmin from scooting to hopping and the DHLA-SB enhanced the turnover rates with thrombin and trypsin by approximately an order of magnitude relative to GSH ligands. The new insights from the broad scope of this study provide an important framework for designing optimized QD conjugates as probes and sensors for enzyme activity.

Human Neutrophil Elastase Activated Fluorescent Probe for Pulmonary Diseases Based on Fluorescence Resonance Energy Transfer Using CdSe/ZnS Quantum Dots

“…Although the overall catalytic efficiency ( k cat / K m ) for QDP is almost identical to the commercial peptide substrate NMeOSuc-AAPV-pNA, the binding affinity for QDP is about 275-fold better than that of the commercial peptide. Such enhanced activity may be due to a so-called “hopping” model of activity, in which the enzyme turns over all of the substrates bound to a particular nanoparticle before diffusing to the next nanoparticle–substrate conjugate. − Our further experiment confirmed that the elevated binding affinity for HNE toward QDP was independent of the ratio of QD with peptide (Table S3). These observations suggested that QDP was a promising substrate for HNE enzymatic study.…”

Transducing Protease Activity into DNA Output for Developing Smart Bionanosensors

“…Nevertheless, examining the resulting data profiles in Figure showed that, despite this leakage, the activity of the individual proteases, both separately and together, could still be discriminated from each other by the unique signal pattern generated. The leakage noted in this instance may reflect DNA interactions with the QD surface which is known to be complex, somewhat unpredictable, and has the ability to bind to many types of biologicals in a nonspecific manner even when displaying PEGylated ligands . One aspect of future work will focus on designing an all‐nucleic acid version of this sensor to potentially try to address this issue.…”

Quantum Dots and Gold Nanoparticles as Scaffolds for Enzymatic Enhancement: Recent Advances and the Influence of Nanoparticle Size

“…Lysozyme was assembled on 2.5 and 6.3 nm diameter CdSe QDs and investigated for resulting conformational changes depending on QD size and whether the QDs impacted lysozyme activity (Figure 7a). Their results showed that conformational changes in lysozyme occurred for both QDs and that the smaller, higher curvature 2.5 nm QDs were found to promote more protein α-helical structure (via circular dichroism) and greater enzymatic activity-here, lysis of Gram-positive bacterium Micrococcus lysodeikticus-compared to larger QDs [108] (Figure 7b). Specifically, increasing the 2.5 nm QD concentration from 20 nM to 50 nM decreased the percent helix content of lysozyme from 32.89% to 24.09%.…”