Abstract
We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity.
Highlights
Protein kinases have been recognized as one of the largest families of cell-regulatory molecules with more than 500 encoded in the human genome [1,2]
As depicted in Scheme 1, once a dye-tethered peptide substrate is phosphorylated by a protein kinase, the addition of Zn(II) may lead to a strong fluorescent resonance energy transfer (FRET) signal between the quantum dots (QDs) as an energy donor and the dye as an energy acceptor, whereas the unphosphorylated peptides may not cause the FRET
While divalent metal ions (Ni(II), Co(II), Cu(II), and Zn(II)) and a trivalent metal ion (Fe(III)) were tested, only Zn(II) ion triggered a strong association between the energy donor and acceptor of the quantum dot-fluorescent resonance energy transfer (QD-FRET) in the presence of T-pPEP1 (Figure 1a and b)
Summary
Protein kinases have been recognized as one of the largest families of cell-regulatory molecules with more than 500 encoded in the human genome [1,2]. Since Zn(II) naturally has a strong interaction with phosphate ions in zinc-binding enzymes [22,23], and is well-coordinated with multidentate ligands based on metal binding affinity [24], we reason that phosphopeptides may be preferably associated via Zn(II) coordination with the functional groups (i.e., carboxyl groups) of a QD surface that may act as multidentate ligands. This improved association can be realized, based on the increased surface density of functional groups due to the large surface-to-volume ratio of the nanoparticles. We envisage that QD-FRET could be an alternative to avoid this issue
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