Elucidating Surface Ligand-Dependent Kinetic Enhancement of Proteolytic Activity at Surface-Modified Quantum Dots

Abstract

Combining biomolecules such as enzymes with nanoparticles has much to offer for creating next generation synergistically functional bionanomaterials. However, almost nothing is known about how these two disparate components interact at this critical biomolecular-materials interface to give rise to improved activity and emergent properties. Here we examine how the nanoparticle surface can influence and increase localized enzyme activity using a designer experimental system consisting of trypsin proteolysis acting on peptide-substrates displayed around semiconductor quantum dots (QDs). To minimize the complexity of analyzing this system, only the chemical nature of the QD surface functionalizing ligands were modified. This was accomplished by synthesizing a series of QD ligands that were either positively or negatively charged, zwitterionic, neutral, and with differing lengths. The QDs were then assembled with different ratios of dye-labeled peptide substrates and exposed to trypsin giving rise to progress curves that were monitored by Förster resonance energy transfer (FRET). The resulting trypsin activity profiles were analyzed in the context of detailed molecular dynamics simulations of key interactions occurring at this interface. Overall, we find that a combination of factors can give rise to a localized activity that was 35-fold higher than comparable freely diffusing enzyme-substrate interactions. Contributing factors include the peptide substrate being prominently displayed extending from the QD surface and not sterically hindered by the longer surface ligands in conjunction with the presence of electrostatic and other productive attractive forces between the enzyme and the QD surface. An intimate understanding of such critical interactions at this interface can produce a set of guidelines that will allow the rational design of next generation high-activity bionanocomposites and theranostics.

Transducing Protease Activity into DNA Output for Developing Smart Bionanosensors

“…We note that trypsin's kinetic activity on the QD‐PD substrate appears to be slightly slower (as visualized by comparison of the slope of kinetic data) than that previously noted with other QD‐peptide substrates (no DNA adduct) . We hypothesize that this may be attributable to electrostatic attraction and/or steric repulsion between the trypsin and the DNA . As detailed previously, placing a peptide substrate for trypsin on similarly functionalized QDs, resulted in up to fivefold increases in apparent catalytic activity .…”

“…The upstream sensing assembly for the chymotrypsin sensor configuration was tested in a similar manner and manifested similar results albeit requiring more chymotrypsin because of that enzyme's lower specific activity (see Figure S7, Supporting Information). We note that trypsin's kinetic activity on the QD‐PD substrate appears to be slightly slower (as visualized by comparison of the slope of kinetic data) than that previously noted with other QD‐peptide substrates (no DNA adduct) . We hypothesize that this may be attributable to electrostatic attraction and/or steric repulsion between the trypsin and the DNA .…”

“…The peptide sequences are somewhat modular and in overall design are similar to previous proteolytic substrates designed for use with QDs . The hexahistidine sequence, (His) 6 , functions as a QD attachment module since it rapidly coordinates to the Zn on the QD's shell with high affinity ( K d ≈ 1 × 10 −9 M) so that conjugation comes about simply by mixing the PDs with the QDs . Moreover, this self‐assembly occurs in a ratiometric manner providing for control over the number of peptides per QD.…”

“…Contiguous with the (His) 6 , each of the peptides contains a Ser residue followed by a helical (Ala) 3 motif, and these provide flexibility so that the peptide can extend out away from the QD surface and display the specific protease cleavage sequences. The presence of surface ligand on the QD surface will help accommodate and promote an extended configuration by not allowing the peptide to settle or fold around the QD . The terminal cysteine (Cys) residues provide unique thiol groups that are used in this context as a chemoselective handle for linkage to aminolated DNA via a heterobifunctional succinimidyl‐4‐[ N ‐maleimidomethyl]cyclohexane‐1‐carboxylate (SMCC) cross‐linker.…”

“…The ability to self‐assemble and arrange multiple acceptors around each QD donor allows us to controllably tune FRET efficiency ( E FRET ), see the Experimental Section. In the context of these sensors, being able to predetermine the number of dye acceptors displayed on the QD allows us to optimize the FRET response following proteolysis . To implement this, we began by characterizing the FRET properties of the 525 QD donors as increasing ratios of Cy3‐acceptor‐labeled Trypsin PDs were assembled onto them.…”