Sensing caspase 3 activity with quantum dot-fluorescent protein assemblies

Abstract

We demonstrate the use of a hybrid fluorescent protein semiconductor quantum dot (QD) sensor capable of specifically monitoring caspase 3 proteolytic activity. mCherry monomeric red fluorescent protein engineered to express an N-terminal caspase 3 cleavage site was ratiometrically self-assembled to the surface of QDs using metal-affinity coordination. The proximity of the fluorescent protein to the QD allows it to function as an efficient fluorescence resonance energy transfer acceptor. Addition of caspase 3 enzyme to the QD-mCherry conjugates specifically cleaved the engineered mCherry linker sequence, altering the energy transfer with the QD and allowing quantitative monitoring of proteolytic activity. Inherent advantages of this sensing approach include bacterial expression of the protease substrate in a fluorescently appended form, facile self-assembly to QDs, and the ability to recombinantly modify the substrate to target other proteases of interest.

Figure 1

(A) Schematic of the QD-fluorescent protein sensor. mCherry with an N-terminal linker expressing the caspase 3 cleavage site and a His₆ sequence were self-assembled to the surface of CdSe-ZnS DHLA-QDs resulting in FRET quenching of the QD and sensitized emission from the mCherry-acceptor (mCherry PDB structure 2h5q). Caspase 3 cleaves the linker reducing FRET efficiency. (B) Linker sequences. The original 35 residue N-terminal linker is shown with colors highlighting functionalities including the start methionine (Met), His₆ NTA-purification/QD assembly sequence, several other functionalities, and the first residues of the mCherry protein. Caspase 3 cleavage sites insertions into the linker are shown in yellow.

Figure 2

(A) Deconvoluted spectra from 550 nm QDs (quantum yield 0.2) self-assembled with an increasing ratio of mCherry substrate 1. Data are corrected for mCherry direct excitation. (B) Plots of normalized QD PL vs. mCherry ratio or valence n (red), FRET efficiency (blue), and efficiency corrected for heterogeneity using a Poisson distribution function (green).

Figure 3

Proteolytic velocity vs. substrate concentration. Changes in FRET efficiency were converted to enzymatic velocity (nM mCherry cleaved/min) as described. Representative data are shown for linker 1 (A) and extended linker 2 (B) substrates (3.5 and 1.5 mCherry/QD valence utilized, respectively) along with estimated K_M and V_max values (mean of 3 experiments). Data were fit with Eq 2.