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Fluorescence resonance energy transfer in quantum dot-protein kinase assemblies.

Yildiz I, Gao X, Harris TK, Raymo FM - J. Biomed. Biotechnol. (2007)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Supramolecular Science, Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146-0431, USA.

ABSTRACT
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.

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Emission spectra of CdSe-ZnS core-shell QDs (0.1 μM inborate buffer, pH = 7.4, T = 20°C, λEX = 442 nm) coated with DHLA in the absence (a) and presence of 0.5 (b), 1.0 (c), 2.0 (d), 5.0 (e), and 10.0 (f) μM of His6-PDK1(ΔPH).
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fig1: Emission spectra of CdSe-ZnS core-shell QDs (0.1 μM inborate buffer, pH = 7.4, T = 20°C, λEX = 442 nm) coated with DHLA in the absence (a) and presence of 0.5 (b), 1.0 (c), 2.0 (d), 5.0 (e), and 10.0 (f) μM of His6-PDK1(ΔPH).

Mentions: The emission spectrum (a in Figure 1) of CdSe-ZnS core-shell QDs coatedwith DHLA in borate buffer (pH = 7.4) shows an intense band centered at 600 nmwith a quantum yield of 0.2. Theaddition of His6-PDK1(ΔPH) to this dispersion affects theemissive behavior of the nanoparticles. Specifically,the luminescence increases with the concentration of His6-PDK1(ΔPH)(a–f in Figure 1) in agreement with the adsorption of the protein on the surface ofthe QDs. Indeed, literature reports [6] demonstrate that the coating of CdSe-ZnS core-shell QDs withhistidine-tagged proteins leads to a luminescence enhancement, as a result ofthe significant change in the local environment around the emissive inorganicparticles. In particular, the plot (see Figure 2) of the emissionintensity of our QDs at 600 nm against the relative concentration of His6-PDK1(ΔPH) showsthat saturation is reached at a protein/QD ratio of ca. 30. Under these conditions, the luminescencequantum yield of the nanoparticles is 0.4.


Fluorescence resonance energy transfer in quantum dot-protein kinase assemblies.

Yildiz I, Gao X, Harris TK, Raymo FM - J. Biomed. Biotechnol. (2007)

Emission spectra of CdSe-ZnS core-shell QDs (0.1 μM inborate buffer, pH = 7.4, T = 20°C, λEX = 442 nm) coated with DHLA in the absence (a) and presence of 0.5 (b), 1.0 (c), 2.0 (d), 5.0 (e), and 10.0 (f) μM of His6-PDK1(ΔPH).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279162&req=5

fig1: Emission spectra of CdSe-ZnS core-shell QDs (0.1 μM inborate buffer, pH = 7.4, T = 20°C, λEX = 442 nm) coated with DHLA in the absence (a) and presence of 0.5 (b), 1.0 (c), 2.0 (d), 5.0 (e), and 10.0 (f) μM of His6-PDK1(ΔPH).
Mentions: The emission spectrum (a in Figure 1) of CdSe-ZnS core-shell QDs coatedwith DHLA in borate buffer (pH = 7.4) shows an intense band centered at 600 nmwith a quantum yield of 0.2. Theaddition of His6-PDK1(ΔPH) to this dispersion affects theemissive behavior of the nanoparticles. Specifically,the luminescence increases with the concentration of His6-PDK1(ΔPH)(a–f in Figure 1) in agreement with the adsorption of the protein on the surface ofthe QDs. Indeed, literature reports [6] demonstrate that the coating of CdSe-ZnS core-shell QDs withhistidine-tagged proteins leads to a luminescence enhancement, as a result ofthe significant change in the local environment around the emissive inorganicparticles. In particular, the plot (see Figure 2) of the emissionintensity of our QDs at 600 nm against the relative concentration of His6-PDK1(ΔPH) showsthat saturation is reached at a protein/QD ratio of ca. 30. Under these conditions, the luminescencequantum yield of the nanoparticles is 0.4.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Supramolecular Science, Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146-0431, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus