Quantum Dot–Peptide Conjugates as Energy Transfer Probes for Sensing the Proteolytic Activity of Matrix Metalloproteinase-14

Abstract

We detail the assembly and characterization of quantum dot (QD)−dye conjugates constructed using a peptide bridge specifically designed to recognize and interact with a breast cancer biomarkermatrix metalloproteinase-14 (MMP-14). The assembled QD conjugates are then used as optically addressable probes, relying on Förster resonance energy transfer (FRET) interactions as a transduction mechanism to detect the activity of MMP-14 in solution phase. The QDs were first coated with dithiolane poly(ethylene glycol) (PEG) bearing a carboxyl group that allows coupling via amide bond formation with different dye-labeled peptides. The analytical capability of the conjugates is enabled by correlating changes in the FRET efficiency with the conjugate valence and/or QD-to-dye separation distance, triggered and modulated by enzymatic proteolysis of surface-tethered peptides. The FRET probe exhibits great sensitivity to enzyme digestion with sub-nanomolar limit of detection. We further analyze the proteolysis data within the framework of the Michaelis–Menten model, which considers the fact that surface-attached peptides have a slower diffusion coefficient than free peptides. This results in reduced collision frequency and lower catalytic efficiency, k cat/K M. Our results suggest that our conjugate design is promising, effective, and potentially useful for in vivo analysis.

Endoproteolysis of Oligopeptide-Based Coacervates for Enzymatic Modeling

“…Our findings are not unusual because enzyme kinetics on a scaffold (e.g., particle or membrane) have been a topic of debate, with some supporting a slow kinetic model that considers slow diffusion and steric crowdedness, , while others favor a fast kinetic model that emphasizes enzyme hopping and high local concentration . On the one hand, our finding agrees with a recent study by Keating et al, showing that phosphatase exhibits slightly higher activity toward substrates within coacervates compared to free substrates, and kinase activity was not significantly altered by the presence of coacervates .…”

Evaluating the Catalytic Efficiency of the Human Membrane-type 1 Matrix Metalloproteinase (MMP-14) Using AuNP–Peptide Conjugates

“…50- to 180-fold reduction compared to free substrates in solution. We would like to note that the reduction in the enzyme catalytic efficiency (namely for MMP-14) was also measured using peptides that have been covalently coupled to luminescent PEG-stabilized QDs combined with changes in the fluorescence resonance energy transfer (FRET) interactions …”

“…We would like to note that the reduction in the enzyme catalytic efficiency (namely for MMP-14) was also measured using peptides that have been covalently coupled to luminescent PEG-stabilized QDs combined with changes in the fluorescence resonance energy transfer (FRET) interactions. 58 We ascribe the reduction in the catalytic efficiency measured for the enzyme using our conjugates to the sample configuration. A more appropriate interpretation should consider a rigorous evaluation of the biomolecular activities for free versus NP-tethered peptides.…”

Ligation of Gold Nanoparticles with Self-Assembling, Coiled-Coil Peptides

“…28 To rule out the fact that the emission of Cy3K25 was nonspecifically quenched by Cy5 (i.e., that Cy3K25 is not quenched by Cy5 alone), we performed a control experiment where Cy3K25 was titrated with increasing amounts of free sulfonated Cy5: No quenching at 568 nm was observed (Figure S5a,b). 29 The PE reaction was performed in a 384-well plate using 300 nM Cy3K25, 450 nM Cy5E21, and increasing concentrations of E25 (Figure 1c). Fluorescence at 568 nm was turned back on with increasing concentrations of E25, thus indicating an increased distance from Cy3K25 to Cy5E21 and disruption of their CC morphology.…”