Quantum dots (QDs) have been increasingly used in biolabeling recently as their advantages over molecular fluorophores have become clear. For bioapplications QDs must be water-soluble and buffer stable, making their synthesis challenging and time-consuming. A simple aqueous synthesis of silica-capped, highly fluorescent CdTe quantum dots has been developed. CdTe QDs are advantageous as the emission can be tuned to the near-infrared where tissue absorption is at a minimum, while the silica shell can prevent the leakage of toxic Cd 2+ and provide a surface for easy conjugation to biomolecules such as proteins. The presence of a silica shell of 2-5 nm in thickness has been confirmed by transmission electron microscopy and atomic force microscopy measurements. Photoluminescence studies show that the silica shell results in greatly increased photostability in Tris-borate-ethylenediaminetetraacetate and phosphate-buffered saline buffers. To further improve their biocompatibility, the silica-capped QDs have been functionalized with poly(ethylene glycol) and thiol-terminated biolinkers. Through the use of these linkers, antibody proteins were successfully conjugated as confirmed by agarose gel electrophoresis. Streptavidin-maleimide and biotinylated polystyrene microbeads confirmed the bioactivity and conjugation specificity of the thiolated QDs. These functionalized, silica-capped QDs are ideal labels, easily synthesized, robust, safe, and readily conjugated to biomolecules while maintaining bioactivity. They are potentially useful for a number of applications in biolabeling and imaging.

PrP binds copper in the highly conserved, unstructured N-terminal half of the protein. The octarepeat region consists of 4 tandem repeats of PHGGGWGQ and binds four equivalents of copper at full occupancy. Adjacent to the octarepeats are two additional histidines that may also bind copper. We recently showed that when the octarepeat region is titrated with Cu 2+ , the copper binding mode depends on the number of equivalents of copper bound. In addition to copper, other metals have been associated with PrP, however zinc is the only metal other than copper that induces PrP endocytosis, inhibits fibril formation and promotes inter-molecular interactions. In this work we show that even large excesses of zinc (> 1mM) are unable to displace copper from either the octarepeat region or the full-length protein. However, EPR reveals that physiologically relevant levels of zinc significantly alter the distribution of copper among the available binding modes. Diethyl pyrocarbonate (DEPC) modification and Mass Spectrometry is used to identify the octarepeat region as the zinc binding site and to confirm that the affinity of PrP for zinc is ~200 μM. PrP can simultaneously bind both copper and zinc by shifting to binding modes that minimize the ratio of histidines to copper.

Current research suggests that the function of the prion protein (PrP) is linked to its ability to bind copper. PrP is implicated in copper regulation, copper buffering and copper-dependent signaling. Moreover, in the development of prion disease, copper may modulate the rate of protein misfolding. PrP possesses a number of copper sites, each with distinct chemical characteristics. Most studies thus far have concentrated on elucidating chemical features of the octarepeat region (residues 60-91, hamster sequence), which can take up to four equivalents of copper, depending on the ratio of Cu2+ to protein. However, other sites have been proposed, including those at histidines 96 and 111, which are adjacent to the octarepeats, and also at histidines within PrP's folded C-terminal domain. Here, we review the literature of these copper sites extrinsic to the octarepeat region and add new findings and insights from recent experiments.