应用于蛋白质结构测定的新型顺磁探针探索

Paramagnetic nuclear magnetic resonance (NMR) spectroscopy has been recognized as powerful tools for the characterization of protein structures and their dynamics and interactions in solution. Paramagnetic probe, providing valuable angular and long-distance restraints for structure calculations, is the most important component in the paramagnetic system. Paramagnetic lanthanoids are the most frequently used probes as chemical shift mediators and line broadening agents, but, still exist shortcomings and deficiencies. At First, only few probes are applicable in living cells since most tags showing good performances in solution are easy to detach from protein and release lanthanoids in presence of intracellular reductants and other metal ions. Secondly, two or more probes, and mutant sites are needed to get the structure information of the whole protein, increasing much workload and complexity of protein study. At last, the spin- and fluorescence-labeling and detection are conducted separately, which makes it hard to build connections of information obtained from NMR and fluorescence assays. Herein, novel paramagnetic lanthanoid tags for protein study in living cells are designed and synthetized to settle these problems above. We apply photo-induced isomerization to get more restraints from one probe to improve the simplicity and accuracy of protein study and employ a general approach of photo-induced luminescence with small organic fluorescent pigments to label the protein with spin and fluorescence at once, which could elucidate the structure and function of protein at atomic resolution and reveal transportation, distribution and degradation of protein at the cellular level at the same time. Our work will not only simplify the procedure but also get a better understanding of the function and mechanism of protein, further accelerating the development of this area.

顺磁核磁共振技术是研究溶液状态下蛋白质结构和动力学的最重要手段之一。其中顺磁探针提供距离和角度约束信息，是该技术的核心组成部分。目前研究最广泛的镧系顺磁探针存在以下问题：适用于细胞的探针少，在溶液中性能优异的探针难以在复杂的细胞环境下工作；需要参比或同类型的多种探针，经过多个突变位点的探索才能够获取目标蛋白的全部结构信息，流程繁琐；蛋白质需分别进行顺磁和荧光标记，无法同时、准确反映蛋白质顺磁和荧光信息之间的关系。因此，发展新型顺磁探针仍是该领域亟待解决的问题之一。本申报项目拟设计合成适用于活细胞的顺磁探针，利用光致变构原理实现一个探针获取多套顺磁数据方法；连接普适荧光基团实现顺磁探针发光，同时观察蛋白质原子尺度的构象变化和细胞水平的转运，定位及降解等过程。该申报项目既简化了实验过程，又可以更好地解析蛋白质功能和作用机制，有望取得较为前沿的成果，进一步推动细胞内蛋白质的研究。

本项目旨在设计合成用于活细胞内的新型顺磁探针，同时具有光响应单元，以期获取蛋白质更多的结构和功能信息。项目在执行过程中基本完成了设定目标。一是通过细胞内硫醇小分子检测实验，系统性地研究了砜基与巯基的位点特异性标记反应，发现合适的拉电子取代基是保证连接单元反应快速、专一、高效的关键因素。实验还利用19F NMR探针实现了近20种结构相似的小分子硫醇的“二维码”绘制；细胞内小分子的硫醇的识别；谷胱甘肽不可逆加成之后的代谢途径的解析；以含硫醇基团为底物的酶活性检测（γ-谷氨酰转肽酶）。二是在上述基础之上，设计合成了反应活性高、构象单一的镧系顺磁标签MSQu-DOTA-Ln。标签采用了新的催化方法在室温下高效合成了砜基化合物，成功规避了金属高温络合引起的砜基水解和硫醚氧化低产率等合成方法带来的问题。该标签可以在近生理条件下与蛋白质的半胱氨酸残基形成稳定的硫醚键，连接效率接近100%，并且产生较大的赝接触位移和残余偶极耦合效应。标签进一步在非洲爪蟾卵母细胞中验证了其良好的稳定性和顺磁特性，将有效地推动细胞内顺磁核磁共振的研究。其中，金属铕(Ⅲ)配合物发出明亮的红光，可同时作为发光标记，获取蛋白质在细胞层次的信息。