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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 mixture ofCdSe-ZnS core-shell QDs (0.1 μM),His6-PDK1(ΔPH) (10 μM), BODIPY-ATP (10 μM)and ATP (301 μM) (a) CdSe-ZnS core-shell QDs (0.1 μM) (d)and BODIPY-ATP (10 μM) (e)in borate buffer (pH = 7.4, T = 20°C, λEX = 442 nm). Deconvolution (b and c)of trace a.
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fig7: Emission spectra of a mixture ofCdSe-ZnS core-shell QDs (0.1 μM),His6-PDK1(ΔPH) (10 μM), BODIPY-ATP (10 μM)and ATP (301 μM) (a) CdSe-ZnS core-shell QDs (0.1 μM) (d)and BODIPY-ATP (10 μM) (e)in borate buffer (pH = 7.4, T = 20°C, λEX = 442 nm). Deconvolution (b and c)of trace a.

Mentions: In principle, the addition of a ligand able to displace BODIPY-ATP from the complementary recognition site of the His6-PDK1(ΔPH)-QD conjugate should result in the physical separation of the nanoparticle donor from theBODIPY acceptor and, therefore, suppress the energy transfer processes (b in Figure 4). Consistently, the additionof increasing amounts of ATP to a solution of the complex formed between BODIPY-ATP and His6-PDK1(ΔPH)-QD causes a decrease in emission intensity with a concomitant hypsochromic shift (a–f in Figure 6). Instead, the titration of a dispersion of the QDs (0.1 μM) with ATP (0–30 μM) has no influence on their emission spectrum. The deconvolution of thefinal spectrum (a in Figure 7) shows the observed emission to be the sum of twodistinct bands (b and c in Figure 7). One of them (b in Figure 7) is centered at 600 nm, corresponds to the emission of the QDs, and its intensity is significantly smaller than that recorded for a dispersion of the QDs alone (d in Figure 7) under otherwise identical conditions. The other band (c in Figure 7) iscentered at 624 nm, corresponds to the emission of the BODIPY dyes, and its intensity is greater thanthat recorded for BODIPY-ATP alone (e in Figure 7) under otherwise identical conditions. Thus, the QDs recover their luminescence onlyin part, even after the addition of a relatively large amount of ATP, and stillsensitize the emission of the BODIPY dyes under these conditions. These observations suggest that a fraction ofthe BODIPY-ATP conjugates remainsassociated with the QDs even in the presence of an excess of ATP (c and d in Figure 4). In agreement with thisinterpretation, the addition of increasing amounts of BODIPY-ATP to adispersion of QDs leads to the disappearance of the nanoparticle emission at600 nm with the concomitant appearance of the BODIPY emission at 624 nm (a–e in Figure 8). Hence, the BODIPY-ATP can acceptthe excitation energy of the QDs despite the absence of the His6-PDK1(ΔPH)coating around the nanoparticles. These resultssuggest that the BODIPY-ATP conjugate canadsorb nonspecifically on the QDs, presumably, as a result of interactions between the chromophoric component and the surface of the nanoparticles.


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 mixture ofCdSe-ZnS core-shell QDs (0.1 μM),His6-PDK1(ΔPH) (10 μM), BODIPY-ATP (10 μM)and ATP (301 μM) (a) CdSe-ZnS core-shell QDs (0.1 μM) (d)and BODIPY-ATP (10 μM) (e)in borate buffer (pH = 7.4, T = 20°C, λEX = 442 nm). Deconvolution (b and c)of trace a.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279162&req=5

fig7: Emission spectra of a mixture ofCdSe-ZnS core-shell QDs (0.1 μM),His6-PDK1(ΔPH) (10 μM), BODIPY-ATP (10 μM)and ATP (301 μM) (a) CdSe-ZnS core-shell QDs (0.1 μM) (d)and BODIPY-ATP (10 μM) (e)in borate buffer (pH = 7.4, T = 20°C, λEX = 442 nm). Deconvolution (b and c)of trace a.
Mentions: In principle, the addition of a ligand able to displace BODIPY-ATP from the complementary recognition site of the His6-PDK1(ΔPH)-QD conjugate should result in the physical separation of the nanoparticle donor from theBODIPY acceptor and, therefore, suppress the energy transfer processes (b in Figure 4). Consistently, the additionof increasing amounts of ATP to a solution of the complex formed between BODIPY-ATP and His6-PDK1(ΔPH)-QD causes a decrease in emission intensity with a concomitant hypsochromic shift (a–f in Figure 6). Instead, the titration of a dispersion of the QDs (0.1 μM) with ATP (0–30 μM) has no influence on their emission spectrum. The deconvolution of thefinal spectrum (a in Figure 7) shows the observed emission to be the sum of twodistinct bands (b and c in Figure 7). One of them (b in Figure 7) is centered at 600 nm, corresponds to the emission of the QDs, and its intensity is significantly smaller than that recorded for a dispersion of the QDs alone (d in Figure 7) under otherwise identical conditions. The other band (c in Figure 7) iscentered at 624 nm, corresponds to the emission of the BODIPY dyes, and its intensity is greater thanthat recorded for BODIPY-ATP alone (e in Figure 7) under otherwise identical conditions. Thus, the QDs recover their luminescence onlyin part, even after the addition of a relatively large amount of ATP, and stillsensitize the emission of the BODIPY dyes under these conditions. These observations suggest that a fraction ofthe BODIPY-ATP conjugates remainsassociated with the QDs even in the presence of an excess of ATP (c and d in Figure 4). In agreement with thisinterpretation, the addition of increasing amounts of BODIPY-ATP to adispersion of QDs leads to the disappearance of the nanoparticle emission at600 nm with the concomitant appearance of the BODIPY emission at 624 nm (a–e in Figure 8). Hence, the BODIPY-ATP can acceptthe excitation energy of the QDs despite the absence of the His6-PDK1(ΔPH)coating around the nanoparticles. These resultssuggest that the BODIPY-ATP conjugate canadsorb nonspecifically on the QDs, presumably, as a result of interactions between the chromophoric component and the surface of the nanoparticles.

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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