3,4-Dihydroxyphenylalanine Peptides as Nonperturbative Quantum Dot Sensors of Aminopeptidase.

Abstract

Fluorescence-based assays for hydrolases that cleave within the substrate (endopeptidases) are common, while developing substrates for proteases that selectively cleave from peptide termini (exopeptidases) is more challenging, since the termini are specifically recognized by the enzyme and cannot be modified to facilitate a Förster resonance energy transfer (FRET)-based approach. The development of a robust system that enables the quenching of fluorescent particles by simple amino acid side chains would find broad utility for peptide sensors and would be advantageous for exopeptidases. Here we describe a quantum dot (QD)-based electron transfer (ET) sensor that is able to allow direct, quantitative monitoring of both exopeptidase and endopeptidase activity. The incorporation of 3,4-dihydroxyphenylalanine (DOPA) into the sequence of a peptide allows for the quenching of QD photoluminescence through an ET mechanism. DOPA is a nonproteinogenic amino acid that can replace a phenylalanine or tyrosine residue in a peptide sequence without severely altering structural properties, allowing for its introduction at multiple positions within a biologically active peptide substrate. Consequently, the quenching system presented here is ideally suited for incorporation into diverse peptide substrates for enzyme recognition, digestion, and activity sensing. Our findings suggest a broad utility of a small ET-capable amino acid side chain in detecting enzyme activity through ET-mediated QD luminescence quenching.