Department of Engineering 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, RMIT University, 124 La Trobe Street, Melbourne, VIC 3000, Australia.
Australian Synchrotron, 800 Blackburn Road, Clayton, VIC 3168, Australia.
Int J Biol Macromol. 2020 Dec 1;164:3474-3484. doi: 10.1016/j.ijbiomac.2020.08.224. Epub 2020 Aug 31.
Enhanced Green Fluorescent Protein (EGFP) is a biomolecule with intense and natural fluorescence, with biological and medical applications. Although widely used as a biomarker in research, its application as a biosensor is limited by the lack of in-depth knowledge regarding its structure and behavior in adverse conditions. This study is focused on addressing this need by evaluating EGFP activity and structure at different pH using three-dimensional fluorescence, circular dichroism and small-angle X-ray scattering. The focus was on the reversibility of the process to gain insights for the development of biocompatible pH-biosensors. EGFP was highly stable at alkaline pH and quenched from neutral-to-acidic pH. Above pH 6.0, the fluorescence loss was almost completely reversible on return to neutral pH, but only partially reversible from pH 5.0 to 2.0. This work updates the knowledge regarding EGFP behavior in pH by accounting for the recent data on its structure. Hence, it is evident that EGFP presents the required properties for use as natural, biocompatible and environmentally friendly neutral to acidic pH-biosensors.
增强型绿色荧光蛋白（EGFP）是一种具有强烈自然荧光的生物分子，在生物和医学领域有广泛应用。尽管它被广泛用作研究中的生物标志物，但由于缺乏对其在恶劣条件下的结构和行为的深入了解，其作为生物传感器的应用受到限制。本研究旨在通过使用三维荧光、圆二色性和小角 X 射线散射来评估 EGFP 在不同 pH 值下的活性和结构，从而满足这一需求。研究重点是该过程的可逆性，以深入了解开发生物相容性 pH 生物传感器的相关内容。EGFP 在碱性 pH 下高度稳定，在中性到酸性 pH 下被猝灭。在 pH 6.0 以上，当回到中性 pH 时，荧光损失几乎完全可逆，但从 pH 5.0 到 2.0 时仅部分可逆。这项工作通过考虑其结构的最新数据，更新了有关 EGFP 在 pH 值下行为的知识。因此，EGFP 显然具有作为天然、生物相容和环境友好的中性到酸性 pH 生物传感器的所需特性。
