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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 a dispersion of CdSe-ZnS core-shell QDs (0.1 μM)and His6-PDK1(ΔPH) (10 μM) in borate buffer (pH = 7.4, T = 20°C, λEX = 442 nm) in the absence (a) and presence of 1.0 (b), 3.0 (c), 5.0 (d),10.0 (e), 20.0 (f), and 30.0 (g) μM of BODIPY-ATP.
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fig5: Emission spectra of a dispersion of CdSe-ZnS core-shell QDs (0.1 μM)and His6-PDK1(ΔPH) (10 μM) in borate buffer (pH = 7.4, T = 20°C, λEX = 442 nm) in the absence (a) and presence of 1.0 (b), 3.0 (c), 5.0 (d),10.0 (e), 20.0 (f), and 30.0 (g) μM of BODIPY-ATP.

Mentions: The absorption spectrum (a in Figure 3) of BODIPY-ATP shows anintense band for the fluorescent component of this dyad in the visible region. This absorption is positioned in the samerange of wavelengths where our QDs emit (b in Figure 3) with an overlap integral of 5.3·10−13 M−1cm3,suggesting that the BODIPY dye can accept the excitation energy of thesenanoparticles. The overlap integral (J) was calculated from the emission intensity (I) of the quantum dots, the molar extinction coefficient (ε) of the BODIPY dye and the wavelength (λ) using(1)J=∫0∞Iελ4dλ∫0∞Idλ.Thus, the supramolecularassociation of the ATP fragment of the dyad with the His6-PDK1(ΔPH)coating of the QDs can be exploited to bring the BODIPY fluorophore in closeproximity to the nanoparticles and encourage the transfer of energy from theemissive CdSe core to the organic dye (a in Figure 4). Indeed, the addition of increasing amounts of BODIPY-ATP to the His6-PDK1(ΔPH)-QD conjugate alterssignificantly the emission spectrum (a–g in Figure 5). In particular, theemission band of the QDs at 600 nm fades with an increase in the concentrationof BODIPY-ATP. Concomitantly, a second band for the BODIPYfluorophore grows at 624 nm in agreement with the expected transfer of energyfrom the nanoparticles to the organic dye upon excitation.


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 a dispersion of CdSe-ZnS core-shell QDs (0.1 μM)and His6-PDK1(ΔPH) (10 μM) in borate buffer (pH = 7.4, T = 20°C, λEX = 442 nm) in the absence (a) and presence of 1.0 (b), 3.0 (c), 5.0 (d),10.0 (e), 20.0 (f), and 30.0 (g) μM of BODIPY-ATP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Emission spectra of a dispersion of CdSe-ZnS core-shell QDs (0.1 μM)and His6-PDK1(ΔPH) (10 μM) in borate buffer (pH = 7.4, T = 20°C, λEX = 442 nm) in the absence (a) and presence of 1.0 (b), 3.0 (c), 5.0 (d),10.0 (e), 20.0 (f), and 30.0 (g) μM of BODIPY-ATP.
Mentions: The absorption spectrum (a in Figure 3) of BODIPY-ATP shows anintense band for the fluorescent component of this dyad in the visible region. This absorption is positioned in the samerange of wavelengths where our QDs emit (b in Figure 3) with an overlap integral of 5.3·10−13 M−1cm3,suggesting that the BODIPY dye can accept the excitation energy of thesenanoparticles. The overlap integral (J) was calculated from the emission intensity (I) of the quantum dots, the molar extinction coefficient (ε) of the BODIPY dye and the wavelength (λ) using(1)J=∫0∞Iελ4dλ∫0∞Idλ.Thus, the supramolecularassociation of the ATP fragment of the dyad with the His6-PDK1(ΔPH)coating of the QDs can be exploited to bring the BODIPY fluorophore in closeproximity to the nanoparticles and encourage the transfer of energy from theemissive CdSe core to the organic dye (a in Figure 4). Indeed, the addition of increasing amounts of BODIPY-ATP to the His6-PDK1(ΔPH)-QD conjugate alterssignificantly the emission spectrum (a–g in Figure 5). In particular, theemission band of the QDs at 600 nm fades with an increase in the concentrationof BODIPY-ATP. Concomitantly, a second band for the BODIPYfluorophore grows at 624 nm in agreement with the expected transfer of energyfrom the nanoparticles to the organic dye upon excitation.

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