Improvement of Membrane Protein Structure Determination Using Paramagnetic Restraints

项目总结 目前使用的药物中，有超过一半的药物针对完整的膜蛋白(IMPS)，因为它们具有关键作用 在将细胞与其环境联系起来方面发挥作用。例如，血管心外膜物质(BVES)，它具有 与心脏健康有关，是与人类疾病有关的300多个小儿麻痹症之一，但没有 结构数据，以帮助治疗或药物开发。越来越多的小鬼被 作为流行疾病的推定药物靶点，推动了IMP结构研究的必要性 了解蛋白质的功能及其在疾病中的作用，并促进药物开发。尽管如此， IMP结构的实验测定仍然具有挑战性，因为它们只占蛋白质的3.5%左右 目前保存在蛋白质数据库(PDB)中的结构。我们提出了一种混合实验和 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 这使用了ROSETTANMR，这是一个针对具有有限核磁共振限制的IMPS优化的蛋白质结构预测程序。 该方法使用非天然氨基酸(NNAA)的顺磁标记来收集核磁共振顺磁 束缚。为了实现这一点，我们解决了现场定向稀土离子标记(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 用于顺磁标记和同时收集IMP的距离和方向约束。 ROSETANMR将使用从基于镧系元素的标签获得的核磁共振结构限制用于新的IMP 结构预测。我们预计，顺磁约束将把预测引导到低能量和 实验上有效的构象，显著提高了IMP结构的确定。因此，目标2是 改进了仅使用顺磁约束的IMP结构预测。最终，这些经过优化的 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(具有生物重要性的)IMP，加速其实验结构确定。

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.