顺磁标记蛋白质在溶液与细胞内研究中的应用

Paramagnetic effects in structural determination, interactions and dynamics of proteins in vitro and in cells

核磁共振中的顺磁效应,包括顺磁弛豫增强、赝接触位移和残余偶极耦合,是研究生物大分子结构、动态变化与相互作用的重要结构约束条件.定点顺磁标记大分子是获得该类结构约束的重要途径.本文论述了生物核磁中顺磁效应的基本原理,蛋白质定点顺磁标记路线和方法,以及在生物核磁中产生顺磁弛豫增强、赝接触位移和残余偶极耦合等顺磁效应的标签质量评价.在介绍该领域最新重要进展的基础上,本文主要讨论了本课题组通过利用顺磁效应在结构生物学和化学生物学中的工作:多结构域蛋白质中的结构域动态取向分析;非平衡态下低含量、不稳定蛋白质复合物的三维结构测定;赝接触位移测定活细胞内蛋白质的三维结构;顺磁核磁和电子自旋共振在结构生物学研究中的互补性以及应用双电子自旋共振在细胞内高分辨距离测定方法.顺磁定点标记蛋白质等生物大分子是获得该类大分子在近生理状态下高分辨动态结构与变化信息的重要方法,预计该技术会在细胞内研究中得到广泛应用.

Paramagnetic effects are valuable structural restraints in study of structures, dynamics and interactions of proteins by means of NMR spectroscopy. Site-specific tagging of proteins with paramagnetic ions is generally required to achieve these paramagnetic effects. In this review, we first introduce the concept of paramagnetic effects including paramagnetic relaxation enhancement(PRE), pseudocontact shift(PCS) and residual dipolar couplings(RDCs), which are generally used in biomolecular NMR. Thereafter, strategies of site-specific paramagnetic tagging of proteins are outlined, and the paramagnetic effects are evaluated. On the basis of paramagnetic tagging methods described above, the applications of paramagnetic NMR in characterization of multi-domain protein replacement, 3 D structural determination of unstable and low-abundance enzyme intermediate complex in real time condition, and 3 D structural determination of proteins in live cells using PCSs are introduced. NMR optimized paramagnetic tags suitable for achieving high spatial resolution of distances determined by double electron-electron resonance(DEER) are reviewed. In the end, the applications of DEER measurement in characterization of protein structures in cells are demonstrated. Paramagnetic NMR and electron paramagnetic resonance(EPR) in characterization of proteins in cells is shown as a powerful technique in structural biology and chemical biology, and the development of biological compatible methods of protein modifications suitable for NMR and EPR applications are in high demand. In summary, paramagnetic tagging proteins are a powerful tool in elucidation of protein structures, dynamics and interactions in chemical and structural biology and it is surely to find wide applications in cells.