Fluorescence resonance energy transfer in quantum dot-protein kinase assemblies.
In search of viable strategies to identify selective inhibitors of protein kinases, we have designed a binding assay to probe the interactions of human phosphoinositide-dependent protein kinase-1 (PDK1) with potential ligands. Our protocol is based on fluorescence resonance energy transfer (FRET) between semiconductor quantum dots (QDs) and organic dyes. Specifically, we have expressed and purified the catalytic kinase domain of PDK1 with an N-terminal histidine tag [His(6)-PDK1(DeltaPH)]. We have conjugated this construct to CdSe-ZnS core-shell QDs coated with dihydrolipoic acid (DHLA) and tested the response of the resulting assembly to a molecular dyad incorporating an ATP ligand and a BODIPY chromophore. The supramolecular association of the BODIPY-ATP dyad with the His(6)-PDK1(DeltaPH)-QD assembly encourages the transfer of energy from the QDs to the BODIPY dyes upon excitation. The addition of ATP results in the displacement of BODIPY-ATP from the binding domain of the His(6)-PDK1(DeltaPH) conjugated to the nanoparticles. The competitive binding, however, does not prevent the energy transfer process. A control experiment with QDs, lacking the His(6)-PDK1(DeltaPH), indicates that the BODIPY-ATP dyad adsorbs nonspecifically on the surface of the nanoparticles, promoting the transfer of energy from the CdSe core to the adsorbed BODIPY dyes. Thus, the implementation of FRET-based assays to probe the binding domain of PDK1 with luminescent QDs requires the identification of energy acceptors unable to interact nonspecifically with the surface of the nanoparticles.
Yildiz
Ibrahim
I
Center for Supramolecular Science, Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146-0431, USA.
Gao
Xinxin
X
Harris
Thomas K
TK
Raymo
Françisco M
FM
D003287:Contrast Media / Q000737:chemistry*; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D008422:Materials Testing; D015336:Molecular Probe Techniques; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure*; D010316:Particle Size; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000648:ultrastructure; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D045663:Quantum Dots
D003287:Contrast Media; C000631429:PDK1 protein, human; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D017346:Protein Serine-Threonine Kinases