基于蛋白质液晶化定向排列芯片的RDC核磁样品制备新方法研究

测定残余偶极耦合(RDC)辅助溶液中蛋白质结构解析的研究日益增多。一方面，RDC的大小取决于测定的两个原子核间的距离；另一方面，RDCs可以确定溶液中蛋白质的不同二级结构单元或不同结构域之间的相对取向。尽管测定RDCs的核磁共振(NMR)方法和用于获取不同残余程度的介质的研究已很广泛，然而RDC样品制备时存在着操作复杂、耗时和成功率低等问题。针对这些问题，本项目拟建立一种适用于RDC核磁样品制备的蛋白质液晶化定向排列芯片，利用“Y-ear”型非对称微结构混合器使溶液中的蛋白质在RDC液晶相介质中实现快速、均一、稳定和可控的定向排列，建立高效和自动化的NMR/RDC样品制备新方法。该新方法将应用到人体内胰岛素瘤相关蛋白INSM1截短体ZF1-3（包含3个锌指结构域）的RDCs测量。本课题对NMR/RDC样品制备方法的开拓性发展，将进一步推动溶液中生物大分子的NMR结构研究。

Investigations of the residue dipolar coupling (RDC) measurement for protein NMR structure determination have greatly increased. On one hand, the RDC depends on the distance between the two measured nuclei; on the other hand, RDCs from a specific bond can define the relative orientation of two secondary structural elements or two domains in the protein under study. Despite the extensive developments for RDC measurement using NMR spectroscopy and various RDC media designed for protein molecule arrangement, the preparation of the NMR sample for RDC measurement is complex, time-consuming and easy for failure. To solve these problems, a new method is proposed here to develop a liquid crystalline media microfluidic system for the preparation of RDC measurement samples. Using the "Y-ear" type asymmetric microstructure mixer to inject the protein solutions homogenously with a fixed orientation in the RDC liquid crystal phase, this new efficient and automated method is expected to be more rapid，homogeneous, stable and controllable. Furthermore, this new technique will be used to obtain the RDCs from INSM1 ZF1-3, a truncated version of human Insulinoma-associated protein INSM1, which contains three zinc fingers ZF1, ZF2 and ZF3. This pioneering method for sample preparation for RDC measurements will promote NMR structure analysis for macromolecules in solution.