Dos Santos Nathalia Vieira, Saponi Carolina Falaschi, Greaves Tamar Louise, Pereira Jorge Fernando Brandão
Department of Bioprocesses and Biotechnology, School of Pharmaceutical Sciences, São Paulo State University (UNESP) Rodovia Araraquara-Jaú/Km 01 14800-903 Araraquara SP Brazil
School of Science, College of Science, Engineering and Health, Royal Melbourne Institute of Technology 124 La Trobe Street 3000 Melbourne VIC Australia.
RSC Adv. 2019 Jul 24;9(40):22853-22858. doi: 10.1039/c9ra02567g. eCollection 2019 Jul 23.
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.
荧光蛋白在医学和生物学领域作为生物标志物和生物传感器有许多应用。它们的成功很大程度上得益于对首个分离出的荧光蛋白——野生型绿色荧光蛋白（wtGFP）的修饰，这使得改进型变体如增强型绿色荧光蛋白（EGFP）得以开发。关于EGFP的最初报道表明，与在wtGFP中观察到的两种构象相比，该蛋白呈现单一形式和荧光峰。然而，在通过实验确定EGFP的晶体结构后，发现了两种构象，这引发了关于EGFP结构与其光谱特征之间关系的问题。为了解决这一争议，本研究在较低波长和不同条件（不同浓度、pH值和温度）下评估了EGFP的三维荧光光谱，揭示了该蛋白存在第二个荧光峰。可以确认，新峰不是蛋白质固有荧光的反映，也不是三维荧光光谱的假象。研究还表明，第二个峰依赖于pH值，对高温敏感，且与EGFP浓度呈线性相关，证实了新荧光峰与EGFP蛋白结构之间的直接关系。除了揭示EGFP新的荧光峰外，本研究还表明，三维荧光可作为一种强大的技术用于发现其他难以捉摸的荧光团。
