位点特异标记稀土金属离子在蛋白质构象分析中的应用

Structural determination of protein-protein and protein-ligand complexes is extremely important for understanding the foundations of life. Understanding the dynamic properties of proteins is equally necessary to understand their function. Nuclear magnetic resonance spectroscopy (NMR) has been a powerful tool in understanding the structure-function of biological macromolecules.Encoding parmagnetic species in proteins has been shown to greatly enhance the sensitivity of NMR in biological system. Lanthanides metal ions provide rich source of structural restraints of biomolecules including pseudocontact shifts (PCS), residual dipolar couplings (RDCs) and paramagnetic relaxation enhancement (PRE). As proteins rarely bind lanthanides specifically, generation of paramagnetic data usually depends on site-specific tagging with lanthanide-chelating reagents. At present, chemically synthesized tags are almost invariably attached to the protein by formation of one or two disulfide bonds. This precludes the subsequent use of reducing agents or high pH, and makes these constructs incompatible with thiol-containing ligands. The project builds on the applicant's recent discovery that chemically inert and rigid tag can be achieved by virtue of the thiol-ene reaction. The project will focus on site-specifically labeling of proteins with rigid and chemically stable tags. The rigidity of the tag, stability of the tether, and the thermodynamic stability of lanthanide complex will be addressed in tagging procedures. As a consequence, a wide range of diverse applications will be targeted, including: .(1) Measurement of RDCs induced by the molecular alignment effected by the paramagnetic metal ions in a magnetic field. The diversity of available paramagnetic metal ions and of different molecular shapes and charges of the tags will be exploited to create many different alignment tensors. Multiple alignment tensors are a prerequisite for many applications, including ultra-high-resolution 3D structures of proteins in solution and analysis of subdomain mobility. (2) Measurement of PCS induced by the paramagnetism of lanthanide metal ions. PCS are manifested in chemical shift changes that are extremely easy to measure by NMR spectroscopy. They contain long-range (up to 40 ?) structural information which can be used to determine the 3D structures of protein-protein and protein-ligand complexes. Domain motion of proteins will be assessed by PCS and a new approach of PCS analysis will specifically target systems of high molecular weight (> 50 kDa)..In summary, new tools will be delivered in fast characterization of protein-ligand complex and sudomain mobility of proteins by paramagnetic NMR via site-specific labelling of proteins with lanthanide metal ions.

蛋白质是生命有机体的重要组成成分，蛋白质三维结构在其行使功能过程中发挥着重要作用。核磁共振技术是从原子分辨率上了解蛋白质三维溶液结构的唯一方式，但是常规核磁共振在某些情况下较难获得蛋白质-配体复合物结构，特别是在探讨蛋白质内结构域间的相对取向时灵敏度较低。用稀土金属离子位点特异标记蛋白质能解决以上一些关键问题。本课题主要发展和利用稀土金属离子的磁各向异性张量并利用赝接触化学位移来研究溶液中生物大分子的构象和蛋白间的作用方式，为研究生物大分子溶液构象分析提供新方法。具体是通过有机化学合成新型的有机螯合配体、位点特异标记蛋白质并克服以往蛋白修饰的不足,研究不同蛋白质与其目标分子的作用及蛋白质内结构域间的动态变化，建立以顺磁标记来快速获得生物大分子在溶液中的三维结构和研究构象变化的方法；进一步探讨获得大蛋白质溶液结构和多结构域间的动态变化。这对阐明蛋白质结构与功能关系具有重要意义。

蛋白质是生命有机体的重要组成成分，蛋白质三维结构在其行使功能过程中发挥着重要作用。核磁共振技术是从原子分辨率上了解蛋白质三维溶液结构的唯一方式，但是常规核磁共振在某些情况下较难获得蛋白质-配体复合物结构，特别是在探讨蛋白质内结构域间的相对取向时灵敏度较低。本课题主要发展和利用稀土金属离子的磁各向异性张量并利用赝接触化学位移来研究溶液中生物大分子的构象和蛋白间的作用方式，为研究生物大分子溶液构象分析提供新方法。具体是通过有机化学合成新型的有机螯合配体、化学修饰蛋白质以达到刚性设计要求并克服以往蛋白修饰的不足,研究一系列不同蛋白质与其目标分子的作用以及蛋白质内结构域间的动态变化，建立以顺磁标记来快速获得生物大分子在溶液中的三维结构和研究构象变化的方法；进一步探讨获得大蛋白质溶液结构和多结构域间的动态变化。这对阐明蛋白质结构与功能之间的关系有重要意义，并提高核磁共振技术在结构生物学应用中的应用。本项目在实施过程中发展了以稳定C-S键定点修饰蛋白质的方法，这与以往马来酰亚胺衍生物不同，避免了手性中心的产生；发展了超过5个高性能的顺磁探针并应用到活细胞研究中。在本项目发展的顺磁探针的基础上，申请人发展了利用高性能的顺磁探针研究多结构域蛋白在溶液中的取向；利用发展的高稳定、刚性的顺磁探针测定了活细胞内蛋白质的三维结构，为在活细胞水平上以原子分辨水平研究蛋白质的结构、运动和作用方式提供了重要方法。在本项目的资助下，申请人利用发展的高性能稳定探针可以通过顺磁核磁共振测定非平衡态下酶催化中间体的三维结构，由于这类中间体含量低、寿命短，利用普通生物物理技术无法得到精确的三维结构，因此通过PCS快速测定的优点，本项目发展的顺磁标记方法为快速测定瞬态蛋白质复合物构象提供了重要方法。