Fluorescence resonance energy transfer in quantum dot-protein kinase assemblies.
Fluorescence resonance energy transfer in quantum dot-protein kinase assemblies.
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量子点-蛋白激酶组件中的荧光共振能量转移。
DOI：
10.1155/2007/18081
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发表时间：
2007
影响因子：
--
通讯作者：
Raymo,FranciscoM
中科院分区：
文献类型：
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作者：
Yildiz,Ibrahim;Gao,Xinxin;Harris,ThomasK;Raymo,FranciscoM
为了寻找可行的策略来识别蛋白激酶的选择性抑制剂，我们设计了一种结合试验来探测人磷酸肌醇依赖性蛋白激酶-1（PDK 1）与潜在配体的相互作用。 我们的方案是基于半导体量子点（QD）和有机染料之间的荧光共振能量转移（FRET）。 具体而言，我们表达并纯化了具有N末端组氨酸标签的PDK 1的催化激酶结构域[His 6-PDK 1（ΔPH）]。我们已经将该构建体缀合到用二氢硫辛酸（DHLA）包被的CdSe-ZnS核壳量子点上，并测试了所得组装体对掺入ATP配体和BODIPY发色团的分子二联体的响应。 BODIPY-ATP二联体与His 6-PDK 1（ΔPH）-QD组装体的超分子缔合促进能量在激发时从QD转移到BODIPY染料。 ATP的加入导致BODIPY-ATP从与纳米颗粒缀合的His 6-PDK 1（ΔPH）的结合结构域置换。 然而，竞争性结合并不阻止能量转移过程。 使用缺乏His 6-PDK 1（ΔPH）的QD的对照实验表明，BODIPY-ATP二联体非特异性地吸附在纳米颗粒的表面上，促进能量从CdSe核心转移到吸附的BODIPY染料。 因此，实施基于FRET的测定以探测具有发光QD的PDK 1的结合结构域需要鉴定不能与纳米颗粒表面非特异性相互作用的能量受体。
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 [His6‐PDK1(ΔPH)]. 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 His6‐PDK1(ΔPH)‐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 His6‐PDK1(ΔPH) conjugated to the nanoparticles. The competitive binding, however, does not prevent the energy transfer process. A control experiment with QDs, lacking the His6‐PDK1(ΔPH), 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.