如何表征蛋白质瞬态复合物的结构和动态过程对于阐述重要的生命过程具有重要的意义，但是由于瞬态蛋白质复合物的存在时间通常较短并且含量往往较低，获得其三维结构非常困难。发展新的核磁共振技术并能有效获得该类瞬态复合物的三维构象并在此基础上了解其动态学行为，可以深刻了解该类蛋白质的结构-动态-功能的关系，并能揭示生命运动的一些重要过程。本课题中，我们以金黄色葡萄球菌转肽酶Sortase A作为目标蛋白。Sortase A在催化过程中首先与信号肽形成硫酯中间体，随后信号肽与胞外糖肽形成细胞壁。但是硫酯中间体寿命很短，该类三维结构一直没有报道。我们利用顺磁标记的方式定点选择性标记该蛋白。在此基础上，我们尝试利用不同的顺磁探针获得有效的赝接触位移，并以赝接触位移为结构限制性条件计算Sortase A与信号肽形成的硫酯中间体的三维结构，在得到结构的基础上探讨该类酶的动态学与功能关系以及钙离子的作用。

The formation of transient association between a protein and its binding partner plays an important role in accomplishing the physiological functions of a protein. Characterizations of the three-dimensional (3D) structures of transient protein complexes are generally difficult using routine biophysical methods. This is because the structural restraints need to be collected quickly and trapping by crystallization is rarely possible for a protein complex with limited lifetime. In this proposal, we use Sortase A from Staphylococcus aureus as a model protein and attempt to determine the 3D structure of thioacyl intermediate formed by Sortase A and sorting peptide. Srortase A is a transpeptidase that is essential for cell-wall maintenance and expansion in Gram-positive bacteria. It recognizes polypeptides containing the LPXTG motif (where X can be any amino acid) and cleaves the peptide bond between the threonine and glycine residues accompanied by formation of a thioacyl intermediate with the active-site residue Cys184. To date, no detailed structural information is available for the enzyme-substrate intermediate, because the lifetime of this intermediate is limited and hence its concentration in solution is low. The paramagnetic effects including pseudocontact shifts (PCS) and paramagnetic relaxation enhancement (PRE) are obtained via site-specific labeling Sortase A with a rigid lanthanide binding tag. PCS measured by nuclear magnetic resonance (NMR) spectroscopy contain long-range structural restraints of biomolecules and the possibility of determining the structure of the thioacyl intermediate from the PCSs will be explored in this proposal. The detailed interaction of Sortase A, sorting peptide and calcium will be analyzed by paramagnetic NMR spectroscopy, and the effect of calcium in the enzymatic activity will be revisited based on the determined 3D structure of thioacyl intermediate.