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Revealing a new fluorescence peak of the enhanced green fluorescent protein using three-dimensional fluorescence spectroscopy

机译：使用三维荧光光谱揭示增强的绿色荧光蛋白的新荧光峰值

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摘要

Fluorescent proteins have many applications as biomarkers and biosensors in the medical and biological fields. Their success was largely supported by modifications of the first isolated fluorescent protein, the wild-type Green Fluorescent Protein (wtGFP), which allowed the development of improved variants such as the Enhanced GFP (EGFP). The first reports on EGFP indicated that the protein presented a single form and fluorescence peak, in contrast to the two conformations observed in wtGFP. However, after experimental determination of the crystalline structure of EGFP, two conformations were found, generating questions regarding the relationship between EGFP structure and its spectral characteristics. To resolve the controversy, this study evaluated EGFP 3D fluorescence spectra at lower wavelengths and under distinct conditions (different concentrations, pH and temperatures), revealing the existence of a second fluorescence peak for this protein. It was possible to confirm that the new peak was not a reflection of the intrinsic fluorescence of proteins or an artefact from the 3D fluorescence spectroscopy. It was also shown that the second peak is pH dependent, sensitive to high temperatures and linearly related to EGFP concentration, confirming a direct relationship between the new fluorescence peak and EGFP protein structure. In addition to the revelation of the new EGFP fluorescence peak, this study demonstrated that 3D fluorescence can be used as powerful technique in the discovery of other elusive fluorophores.
机译：荧光蛋白在医学和生物领域中具有许多应用作为生物标志物和生物传感器。它们的成功基本上通过修饰第一分离的荧光蛋白，野生型绿色荧光蛋白（WTGFP）来支持，其允许开发改进的变体，例如增强的GFP（EGFP）。关于EGFP的第一个报告表明，与WTGFP中观察到的两个构象相比，蛋白质呈现单个形式和荧光峰。然而，在实验确定EGFP的晶体结构之后，发现了两个构象，为EGFP结构与其光谱特性之间的关系产生了问题。为了解决争议，该研究评估了较低波长的EGFP 3D荧光光谱，并且在不同的条件下（不同浓度，pH和温度），揭示了该蛋白质的第二荧光峰的存在。可以确认新峰值不是从3D荧光光谱中的蛋白质的内在荧光的反射。还表明第二峰是pH依赖性的，对高温敏感，与EGFP浓度线性相关，确认新的荧光峰和EGFP蛋白质结构之间的直接关系。除了新的EGFP荧光峰值的启示之外，该研究表明，3D荧光可作为发现其他难以置信的荧光团的强大技术。

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  • 来源
    《RSC Advances》 |2019年第40期|共6页
  • 作者

    dos Santos Nathalia Vieira; Saponi Carolina Falaschi; Greaves Tamar Louise; Brandao Pereira Jorge Fernando;

  • 作者单位

    Sao Paulo State Univ UNESP Sch Pharmaceut Sci Dept Bioproc &

    Biotechnol Rodovia Araraquara Jau Km 01 BR-14800903 Araraquara SP Brazil;

    Sao Paulo State Univ UNESP Sch Pharmaceut Sci Dept Bioproc &

    Biotechnol Rodovia Araraquara Jau Km 01 BR-14800903 Araraquara SP Brazil;

    RMIT Univ Coll Sci Engn &

    Hlth Sch Sci 124 La Trobe St Melbourne Vic 3000 Australia;

    Sao Paulo State Univ UNESP Sch Pharmaceut Sci Dept Bioproc &

    Biotechnol Rodovia Araraquara Jau Km 01 BR-14800903 Araraquara SP Brazil;

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  • 正文语种 eng
  • 中图分类 化学;
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