NMR结构生物学之不基于NOEs指认的大蛋白质结构解析方法研究。

Probably the biggest hurdle in solving solution NMR structures of large proteins is collecting a sufficient number of tertiary distance restraints derived from nuclear Overhauser enhancements (NOEs), because resonance overlap and slow molecular tumbling make unambiguous assignments of sidechain resonances difficult. The objective of this project is to develop a robust structure determination protocol for large proteins that do not require NOE assignments. Achieving this goal would make solution NMR a much more accessible technology to the Biology community, in particular to researchers interested in obtaining structural information on large water-soluble proteins or membrane proteins. In an earlier study to determine the structure of the mitochondrial uncoupling protein 2 (UCP2)(Nature 2011), a 34 kDa helical, polytopic membrane protein, we found that using residual dipolar couplings (RDCs) to search for small molecular fragments in the Protein Data Bank (PDB) is an effective way to determine local secondary structures. Encouraged by this result, we propose to establish a protocol that combines orientation restraints from RDCs and distance restraints from paramagnetic relaxation enhancement (PRE) measurements. More specifically, we will first design and test algorithms for RDC/CS-based molecular fragment searching and for piecing together these fragments into larger secondary segments. We will then develop methods for tertiary structure determination, which is based on spatially constraining the secondary segments with PRE restraints. The long flexible MTSL used for traditional spin label is the source of large uncertainty associated with PRE-derived distance restraints. We will thus design and test more rigid nitroxide labels for PRE measurements. Finally, we will explore the use of low resolution EM image as spatial constraint for the NMR-derived structural fragments, and apply the method to determine the structure of the p7 cation channel of the Hepatitis C virus (hexamer, 42 kDa). The p7 channel is an interesting case for merging EM and NMR because its size (42 kDa, hexameric) is in the range for EM single-particle averaging and the oligomeric channel also gives a very good NMR spectrum. We believe that developing an effective NMR protocol based on unambiguous data is important for the general application of this technique in structural characterization of large proteins.

在NMR结构生物学领域，急需要一个稳健快速的结构解析方案，其只依赖于非模糊指认的实验数据就可以解析出大蛋白质的空间结构。本项目研究内容主要是受启发于申请人所在的周界文研究组最近利用RDCs的MFR方法和传统PREs距离约束解析出UCP2膜蛋白质的主链结构(34kDa，Nature 2011)。在此基础上设计更严谨的MFR新算法，结合更准确的PREs距离约束和CS实验信息，进一步发展更普适的蛋白质核磁结构解析新方法。该方法将不需要指认NOEs，从而有效地解决NMR结构解析过程中的技术瓶颈。最后，通过结合已有的EM电子密度图数据和HCV p7六聚体的对称性，解析p7离子通道（42kDa）的溶液结构来验证该新方法的可靠性。所以该新方法将有助于进一步把NMR和EM的结构解析过程结合起来。本项目的完成，对于推广NMR技术在结构生物学领域的广泛应用有重要的意义，特别是对于大蛋白质的结构测定将更加可行。

在用液体NMR技术解析蛋白质结构的过程中，最重要的也是最困难的一个步骤就是找到大量的非模糊指认的长程NOEs距离约束。然而，对于分子量较大的蛋白质（大于30 kDa），侧链的完全指认是非常困难的，因为其侧链谱图的脂肪区NMR谱峰重叠会变得非常严重。本项目发展了一种不依赖于NOE的膜蛋白主链结构解析方法（MFR）。通过构建更加多样性的9残基分子片段数据库，以及设计了片段集合收敛性打分函数的新“片段指认”和“段落构建”算法，开发了一种新的NMRLego软件，使MFR算法更具有效率和普适性，最后应用在目标膜蛋白质AAC的结构解析过程中，当前实验结果已经基本完成了项目的预期计划。此外，基于本项目开发出的相关蛋白质核磁新技术和新方法还应用在SCaMC和HCV P7膜蛋白质的结构与功能研究课题中。目前已发表3篇SCI论文，完成情况良好。