基于光致电荷转移的蛋白质荧光传感器

Photo-induced electron transfer (PET) is the main reaction in photosynthesis, and is of great significant to the magnetic sensing and many other biological processes. Based on genetic encoded method, we have incorporated several PET sensors into specific sites of fluorescent proteins. All of these results will facilitate understanding of the process of PET and designing of genetical encoded PET fluorescent sensors. Protein post-translational levels and the concentration changes of various protein markers are directly related to life process. They have become the important basis for disease diagnosis, disease prediction and efficacy assessment. Currently, the detection of protein modification is mainly using of isotope labeling, MS detection, HPLC, IP assay and fluorescent sensors based on small peptides, however, the above assays are rarely can be directly applied for living cells detection. So the development of simpler, more rapid and sensitive method that can be used for PTM detection and quantitative studies has become the hot spot of protein research. Next, fluorescent sensors that can be used as tyrosine sulfonylation, phosphorylation, glycosylation and lysine acetylation fluorescent probes for in vitro and in vivo protein post translation modification (PTM) detections will be developed. The study will break through the limitation of PTM detection nowadays and realize PTM real-time dynamic observation in vivo. This research will provide a powerful tool for the tyrosine kinase (PTK), lysine acetylation and histone deacetylase (Sirt) mechanism studies. Based on the study, new screening methods for PTK and Sirt inhibitors and activators will be expanded.

光致电荷转移（PET）是光合作用中的主要反应，对磁感知等许多生物过程有重要意义。前期研究中，我们通过基因密码子扩展方法，将多种PET探针引入到蛋白质的特定位点，为研究蛋白质中PET机制，及基于PET设计蛋白质荧光探针提供了有力工具。 在此基础上，我们将发展可以用体内外检测酪氨酸磺酸化、磷酸化、糖基化及赖氨酸乙酰化等翻译后修饰（PTM）的荧光探针。PTM修饰酶是细胞信号的主要执行者，已成为重要的药物靶点及疾病诊断、药效评估的重要依据。而目前用于蛋白质PTM的检测手段极少能直接被应用于活细胞检测。因此，发展简单、快速、灵敏的体内外PTM检测及定量的新方法已成为蛋白质研究的热点。 以上探针的发展将突破目前蛋白质修饰检测的局限，实现体内实时动态观察，对酪氨酸激酶（PTK）、赖氨酸乙酰化及去乙酰化酶（Sirt）机理研究提供有力工具。在此基础上，我们将发展PTK，Sirt抑制及激活剂筛选的新方法。

光致电荷转移（PET）是光合作用中的主要反应，对磁感知等许多生物过程有重要意义。.前期研究中，我们通过基因密码子扩展方法，将多种PET 探针引入到蛋白质的特定位点，为研究蛋白质中PET 机制，及基于PET 设计蛋白质荧光探针提供了有力工具。.在此基础上，我们发展可以用于体内外的非天然氨基酸光致电荷转移探针。并通过非天然计算探针的插入实现了提高蛋白酶活力的方法。.我们将萘酚氨基酸（Naphthol-Ala）引入到各种荧光蛋白变体的活性中心，获得了激发和发射波长均红移60 nm以上的突变体。cpsfGFP-66-Naphthol-Ala晶体结构研究揭示了发色团的形成，为发色团红移机理的揭示提供了结构基础。这些蛋白突变体将可能被作为体内成像研究的标记物，增加探测的灵敏度，进而进行深层组织成像或者作为荧光能量共振转移传感器。.设计了一种可以基因编码的光敏蛋白质，基于光致电荷转移机理成功模拟了天然光合作用系统吸收光能，催化二氧化碳还原的功能。