A genetically encoded fluorescent temperature sensor derived from the photoactive Orange Carotenoid Protein
A genetically encoded fluorescent temperature sensor derived from the photoactive Orange Carotenoid Protein
复制标题
DOI：
10.1038/s41598-019-45421-7
复制
发表时间：
2019-06-20
影响因子：
4.6
通讯作者：
Stepanov, Alexey, V
中科院分区：
文献类型：
--
作者：
Maksimov, Eugene G.;Yaroshevich, Igor A.;Stepanov, Alexey, V
代谢反应的不均匀性导致活细胞不同部位的温度分布不均匀。研究细胞过程的正常功能和病理异常的需求要求开发新的可视化方法。在此之前，我们已经证明了35 kda的可光切换橙色类胡萝卜素蛋白（OCP）具有很强的光转化率温度依赖性，并且其三级结构在光激活时发生了显著的结构重排，这使得该蛋白成为纳米级温度传感器。然而，OCP转化率的测定需要测量类胡萝卜素的吸收，这是不适合显微镜。为了解决这个问题，我们将绿色和红色荧光蛋白（TagGFP和TagRFP）融合到OCP结构中，产生光活性嵌合体。在这种嵌合体中，荧光蛋白的电子激发被OCP中的类胡萝卜素有效地淬灭。基于ocp的嵌合体的光激活触发复杂几何结构的重排，允许通过监测荧光强度的变化来测量转化率。这种方法使我们能够确定微环境的局部温度。讨论了未来改进ocp传感器的方向。
The heterogeneity of metabolic reactions leads to a non-uniform distribution of temperature in different parts of the living cell. The demand to study normal functioning and pathological abnormalities of cellular processes requires the development of new visualization methods. Previously, we have shown that the 35-kDa photoswitchable Orange Carotenoid Protein (OCP) has a strong temperature dependency of photoconversion rates, and its tertiary structure undergoes significant structural rearrangements upon photoactivation, which makes this protein a nano-sized temperature sensor. However, the determination of OCP conversion rates requires measurements of carotenoid absorption, which is not suitable for microscopy. In order to solve this problem, we fused green and red fluorescent proteins (TagGFP and TagRFP) to the structure of OCP, producing photoactive chimeras. In such chimeras, electronic excitation of the fluorescent protein is effectively quenched by the carotenoid in OCP. Photoactivation of OCP-based chimeras triggers rearrangements of complex geometry, permitting measurements of the conversion rates by monitoring changes of fluorescence intensity. This approach allowed us to determine the local temperature of the microenvironment. Future directions to improve the OCP-based sensor are discussed.