Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis-Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD-thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.

We present a molecular characterization of metal-affinity driven self-assembly between CdSe−ZnS core−shell quantum dots (QDs) and a series of proteins and peptides appended with various length polyhistidine tags. In particular, we investigated the kinetics of self-assembly between surface-immobilized QDs and proteins/peptides under flow conditions, as well as between freely diffusing QDs and proteins/peptides (solution phase). In the first configuration, QDs were immobilized onto functionalized substrates and then exposed to dye-labeled peptides/proteins. Using evanescent wave excitation, we assessed self-assembly by monitoring the time-dependent changes in the dye fluorescence. In solution, the kinetics of self-assembly was monitored via energy transfer between QDs and dye-labeled proteins/peptides. These measurements allowed determination of the kinetic parameters, including the association and dissociation rates (k on and k off) and the apparent binding constant (K d). We find that self-assembly is rapid with an equilibrium constant K d -1 ≈ 1 nM for solution self-assembly, confirming that metal-affinity interactions provide QD bioconjugates that are functional and stable.

We demonstrate the use of self-assembled luminescent semiconductor quantum dot (QD) -peptide bioconjugates for the selective intracellular labeling of several eukaryotic cell lines. A bifunctional oligoarginine cell penetrating peptide (based on the HIV-1 Tat protein motif) bearing a terminal polyhistidine tract was synthesized and used to facilitate the transmembrane delivery of the QD bioconjugates. The polyhistidine sequence allows the peptide to self-assemble onto the QD surface via metal-affinity interactions while the oligoarginine sequence allows specific QD delivery across the cellular membrane and intracellular labeling as compared to non-conjugated QDs. This peptidedriven delivery is concentration-dependent and thus can be titrated. Upon internalization, QDs display a punctate-like staining pattern in which some, but not all, of the QD signal is co-localized within endosomes. The effects of constant versus limited exposure to QD-peptide conjugates on cellular viability are evaluated by a metabolic specific assay and clear differences in cytotoxicity are observed. The efficacy of using peptides for selective intracellular delivery is highlighted by performing a multicolor QD labeling, where we found that the presence or absence of peptide on the QD surface controls cellular uptake.

The membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 bears the epitopes of two broadly neutralizing antibodies (Abs), 2F5 and 4E10, making it a target for vaccine design. A third Ab, Fab Z13, had previously been mapped to an epitope that overlaps those of 2F5 and 4E10 but only weakly neutralizes a limited set of primary isolates. Here, libraries of Fab Z13 variants displayed on phage were engineered and affinity selected against an MPER peptide and recombinant gp41. A high-affinity variant, designated Z13e1, was isolated and found to be ϳ100-fold improved over the parental Fab not only in binding affinity for the MPER antigens but also in neutralization potency against sensitive HIV-1. Alanine scanning of MPER residues 664 to 680 revealed that N671 and D674 are crucial for peptide recognition as well as for the neutralization of HIV-1 by Z13e1. Ab competition studies and truncation of MPER peptides indicate that Z13e1 binds with high affinity to an epitope between and overlapping with those of 2F5 and 4E10, with the minimal peptide epitope WASLWNWFDITN. Still, Z13e1 remained about an order of magnitude less potent than 4E10 against several isolates of pseudotyped HIV-1. The sum of our molecular analyses with Z13e1 suggests that the segment on the MPER of gp41 between the 2F5 and 4E10 epitopes is exposed on the functional envelope trimer but that access to the specific Z13e1 epitope within this segment is limited. Thus, the ability of MPER-bearing immunogens to elicit potent HIV-1-neutralizing Abs may depend in part on recapitulating the particular constraints that the functional envelope trimer imposes on the segment of the MPER to which Z13e1 binds.

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