EGFP oligomers as natural fluorescence and hydrodynamic standards.
EGFP oligomers as natural fluorescence and hydrodynamic standards.
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DOI：
10.1038/srep33022
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发表时间：
2016-09-13
影响因子：
4.6
通讯作者：
Tóth K
中科院分区：
文献类型：
--
作者：
Vámosi G;Mücke N;Müller G;Krieger JW;Curth U;Langowski J;Tóth K
EGFP寡聚体是荧光蛋白标记生物分子实验的便捷标准。在本研究中，我们对它们的流体动力学和荧光性质进行了表征。用分析超速离心法和荧光相关光谱法测定了EGFP_(1-4)的扩散系数D，得到了83·4…48.2 μm2/S和97.3…54.8 μm~2/S，从单体到四聚体。一个“桶排成行”的模型与沉积数据最为吻合。齐聚物红移了EGFP的发射光谱，吸收没有任何移动。荧光各向异性降低，表明亚基之间的FRET相同。荧光寿命仅略有下降(4%)，表明FRET对非发射状态下的亚基的猝灭作用不明显。FCS测量的D、粒子数和分子亮度依赖于不同亚基中的暗状态和光诱导过程，导致对单体和低聚物不同的光照功率的依赖。由于亚基可能处于“开”(亮)或“关”(暗)状态，FCS确定的表观亮度与单体的表观亮度不成正比。根据其对亚基数目的依赖关系，确定了一个亚基在pH为8时“开启”状态的概率为96%，在pH为6.38时为77%，即质子化增加了暗状态。EGFP寡聚体标准的这些荧光特性可以帮助解释具有生物学意义的寡聚化EGFP融合蛋白的结果。
EGFP oligomers are convenient standards for experiments on fluorescent protein-tagged biomolecules. In this study, we characterized their hydrodynamic and fluorescence properties. Diffusion coefficients D of EGFP1–4 were determined by analytical ultracentrifugation with fluorescence detection and by fluorescence correlation spectroscopy (FCS), yielding 83.4…48.2 μm2/s and 97.3…54.8 μm2/s from monomer to tetramer. A “barrels standing in a row” model agreed best with the sedimentation data. Oligomerization red-shifted EGFP emission spectra without any shift in absorption. Fluorescence anisotropy decreased, indicating homoFRET between the subunits. Fluorescence lifetime decreased only slightly (4%) indicating insignificant quenching by FRET to subunits in non-emitting states. FCS-measured D, particle number and molecular brightness depended on dark states and light-induced processes in distinct subunits, resulting in a dependence on illumination power different for monomers and oligomers. Since subunits may be in “on” (bright) or “off” (dark) states, FCS-determined apparent brightness is not proportional to that of the monomer. From its dependence on the number of subunits, the probability of the “on” state for a subunit was determined to be 96% at pH 8 and 77% at pH 6.38, i.e., protonation increases the dark state. These fluorescence properties of EGFP oligomeric standards can assist interpreting results from oligomerized EGFP fusion proteins of biological interest.
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