Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy.
Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy.
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DOI：
10.1021/acs.jpcc.6b11235
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发表时间：
2017-01-26
期刊：
影响因子：
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通讯作者：
Bain AJ
中科院分区：
文献类型：
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作者：
Blacker TS;Chen W;Avezov E;Marsh RJ;Duchen MR;Kaminski CF;Bain AJ
大多数荧光蛋白表现出多指数荧光衰变，表明存在一个不同的激发态群体。因此，荧光蛋白之间的FRET应该涉及多条能量传递途径。我们最近证明了在3-磷酸肌醇依赖的蛋白激酶1(PDK1)二聚时，EGFP和mCherry(MC)之间的FRET通路是高度受限的。在远离共面和近静态相互作用的几何构型中，由于施主-受体跃迁偶极矩角度的差异，提出了一种基于κ-2取向因子的FRET限制机制。在这里，这是通过FRET到MC产生的谷胱甘肽(谷胱甘肽)和谷胱甘肽S转移酶(GST)与固有的同质和更具流动性的供体俄勒冈州绿488(OG)。基于敏化荧光的转折点，结合施主窗口强度和各向异性测量，对受主窗口强度进行了新的分析，结果表明，对MC发生了不受限制的FRET。然而，供体窗口中的长寿命各向异性衰减分量揭示了其中没有发生FRET的GST-GSH群体，解释了先前GST-GSH关联的定量FRET测量与其接受值之间的差异。这强调了局部供体-受体环境在调节能量传递中的重要性，以及在荧光蛋白FRET的准确定量中执行光谱分辨强度和各向异性衰减测量的必要性。
Most fluorescent proteins exhibit multiexponential fluorescence decays, indicating a heterogeneous excited state population. FRET between fluorescent proteins should therefore involve multiple energy transfer pathways. We recently demonstrated the FRET pathways between EGFP and mCherry (mC), upon the dimerization of 3-phosphoinositide dependent protein kinase 1 (PDK1), to be highly restricted. A mechanism for FRET restriction based on a highly unfavorable κ2 orientation factor arising from differences in donor–acceptor transition dipole moment angles in a far from coplanar and near static interaction geometry was proposed. Here this is tested via FRET to mC arising from the association of glutathione (GSH) and glutathione S-transferase (GST) with an intrinsically homogeneous and more mobile donor Oregon Green 488 (OG). A new analysis of the acceptor window intensity, based on the turnover point of the sensitized fluorescence, is combined with donor window intensity and anisotropy measurements which show that unrestricted FRET to mC takes place. However, a long-lived anisotropy decay component in the donor window reveals a GST-GSH population in which FRET does not occur, explaining previous discrepancies between quantitative FRET measurements of GST-GSH association and their accepted values. This reinforces the importance of the local donor–acceptor environment in mediating energy transfer and the need to perform spectrally resolved intensity and anisotropy decay measurements in the accurate quantification of fluorescent protein FRET.