Improvement of Membrane Protein Structure Determination Using Paramagnetic Restraints

项目摘要 目前使用的药物中有一半以上靶向整合膜蛋白（IMP），因为它们在膜蛋白中起着关键作用。 在连接细胞和环境中发挥作用。例如，血管心外膜物质（BVES），其具有 与心脏健康有关，是300多种与人类疾病有关的IMP之一，但没有 结构数据，以协助治疗或药物开发。越来越多的IMP已经 作为流行性疾病的假定药物靶点，推动了IMP结构研究的需求， 了解蛋白质功能及其在疾病中的作用，并促进药物开发。不过 IMP结构的实验测定仍然具有挑战性，因为它们仅占蛋白质的3.5%左右 蛋白质数据库（Protein Data Bank，PDB）。我们提出了一个混合实验， IMP结构预测的计算方法。在此，我们开发了一个IMP结构预测协议， 使用ROSETTANMR，这是一种针对有限NMR限制的IMP优化的蛋白质结构预测程序。 该方法使用非天然氨基酸（nNAA）的顺磁标记来收集NMR顺磁标记。 束缚为了实现这一目标，我们在一个实验室中解决了定点镧系元素离子标记（SDLL）的挑战。 蛋白质，并确定最佳的标记策略。本提案的目标1是制定议定书 用于顺磁标记并同时收集IMP的距离和方向限制。 从镧系元素标签获得的NMR结构限制将由ROSETTANMR用于IMP从头 结构预测我们希望顺磁约束将指导预测低能量和 实验有效的构象和显着改善IMP结构的测定。因此，目标2是 仅使用顺磁约束改进IMP结构预测。最终，这些优化 协议可以应用于一系列较大的IMP。这里开发的实验技术将是 可转移到其他IMP（具有生物学重要性），加速其实验结构测定。

PROJECT SUMMARY Over half of drugs currently in use target integral membrane proteins (IMPs) because of the critical roles they play in linking the cell to its environment. For example, blood vessel epicardial substance (BVES), which has been linked to cardiac health, is among more than 300 IMPs implicated in human diseases, but with no structural data to assist in therapy or drug development. The increasing number of IMPs that have been implicated as putative drug targets in prevalent diseases drove the need for IMP structural studies to understand protein function and their role in disease, and to facilitate drug development. Nonetheless, the experimental determination of IMP structures remains challenging, as they make up only about 3.5% of protein structures currently housed in the Protein Data Bank (PDB). We propose a hybrid experimental and computational approach for IMP structure prediction. Herein, we develop an IMP structure prediction protocol that uses ROSETTANMR, a protein structure prediction program optimized for IMPs with limited NMR restraints. The approach uses paramagnetic tagging of non-natural amino acids (nNAAs) to collect NMR paramagnetic restraints. To accomplish this, we tackle the challenge of site-directed lanthanide ion labeling (SDLL) in a protein, and determine optimal labelling strategies. Goal 1 of the present proposal is to develop protocols for paramagnetic tagging and simultaneous collection of distance and orientational restraints for IMPs. NMR structural restraints obtained from lanthanide-based tags will be used by ROSETTANMR for IMP de novo structure prediction. We expect that paramagnetic restraints will guide the prediction to low-energy and experimentally valid conformations and significantly improve IMP structure determination. Thus, goal 2 is to improve IMP structure prediction in using only paramagnetic restraints. Ultimately, these optimized protocols can be applied to a range of larger IMPs. The experimental techniques developed here will be transferable to other IMPs (of biological importance) accelerating their experimental structure determination.