Characterization of rare conformational states of proteins by combination of high-pressure X-raycrystallography with high-pressure NMR spectroscopy. Application to the small GproteinRas, its oncogenic mutants and its drug complexes.

通过高压 X 射线晶体学与高压核磁共振波谱相结合来表征蛋白质的稀有构象状态。

• 批准号: 390275479
• 负责人: Professor Dr. Hans Robert Kalbitzer
• 金额: --
• 依托单位: Lehrstuhl für Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/390275479?language=en
• 关键词: Characterization; rare; conformational; states; proteins

High pressure NMR spectroscopy (HPNMR) represents a powerful, still developing method to detect and characterize rare 'excited' conformational states. These states play an essential role in function and folding of proteins. HPNMR provides their thermodynamic parameters but gives structural information with limited resolution only. High pressure macromolecular x-ray crystallography (HPMX) is a complementary new method in protein science that basically can provide highly resolved spatial structures at high pressures. As component of an important and well-characterized signal transduction system we will concentrate on the small G-Protein Ras (rat sarcoma) protein (Ras mutations are found mutated in more than 30 % of all human cancers). It serves as a well-characterized model for method development in the proposed project, but also novel thermodynamic and structural insights are from high medical importance since they may help with the development of allosteric Ras inhibitors. By HPNMR of Ras in complex with the GTP analog GppNHp four different states 1(0), 1(T), 2(T), and 3(T) could be thermodynamically characterized that coexist at ambient pressure. Small compounds that preferentially bind to state 1(T) can allosterically inhibit the effector interaction and thus in principle can suppress the proliferative effect of oncogenic Ras mutants. The main goals of this project are (1) a thorough quantitative analysis of the rare conformational states of H- and K-Ras and their oncogenic mutants in complex with GDP and GppNHp by HPNMR supported by alternative NMR methods such as CEST and relaxation dispersion measurements, (2) to prove that HPMX can efficiently trap high-energy conformers of proteins identified in solution by high-pressure NMR spectroscopy that were identified in solution by HPNMR, (3) to correlate the observed pressure response of NMR parameters with structural changes detected by HPMX, and (4) to show that in principle the obtained high-pressure crystallographic structures can be used to design specific allosteric inhibitors binding to rare states. For showing if the thermodynamically similar state sin crystal and solution are also structurally similar, population-weighted NOESY spectra are back calculated from the X-ray structures and are compared with the experimental solution state NOESY spectra at different pressures in detail. As preliminary experiments show high pressure soaking with small state specific compounds may represent a novel method to access rare conformational states by crystallography. The dynamics under pressure of the different states will be studied by 2D transient P-jump experiments on isotope enriched Ras protein with the high-pressure jump system developed by us. The study will be complemented by relaxation dispersion and chemical exchange saturation transfer (CEST) experiments at different pressures.

高压核磁共振波谱 (HPNMR) 是一种强大的、仍在开发中的方法，用于检测和表征罕见的“激发”构象状态。这些状态在蛋白质的功能和折叠中发挥着重要作用。 HPNMR 提供了它们的热力学参数，但仅提供了分辨率有限的结构信息。高压高分子X射线晶体学（HPMX）是蛋白质科学中的一种补充新方法，基本上可以在高压下提供高分辨率的空间结构。作为重要且已明确表征的信号转导系统的组成部分，我们将重点关注小 G 蛋白 Ras（大鼠肉瘤）蛋白（在所有人类癌症中，超过 30% 的 Ras 突变均存在突变）。它可以作为拟议项目中方法开发的良好表征模型，而且新颖的热力学和结构见解具有很高的医学重要性，因为它们可能有助于变构 Ras 抑制剂的开发。通过 Ras 与 GTP 类似物 GppNHp 复合物的 HPNMR，可以对在环境压力下共存的四种不同状态 1(0)、1(T)、2(T) 和 3(T) 进行热力学表征。优先结合状态 1(T) 的小化合物可以变构抑制效应子相互作用，因此原则上可以抑制致癌 Ras 突变体的增殖作用。该项目的主要目标是 (1) 通过 HPNMR 对 H- 和 K-Ras 的罕见构象状态及其与 GDP 和 GppNHp 复合的致癌突变体进行彻底的定量分析，并得到 CEST 和弛豫色散测量等替代 NMR 方法的支持，（2）证明 HPMX 可以有效捕获溶液中通过高压 NMR 光谱鉴定的蛋白质的高能构象异构体，（3）将观察到的 NMR 参数的压力响应与结构相关联HPMX 检测到的变化，以及（4）表明原则上获得的高压晶体结构可用于设计与稀有态结合的特异性变构抑制剂。为了表明热力学相似状态sin晶体和溶液在结构上是否也相似，从X射线结构反算了布居加权NOESY谱，并与不同压力下的实验溶液态NOESY谱进行了详细比较。初步实验表明，用小态特定化合物进行高压浸泡可能代表了一种通过晶体学获取稀有构象态的新方法。我们将利用我们开发的高压跃迁系统，对同位素富集的Ras蛋白进行二维瞬态P跃迁实验，研究不同状态压力下的动力学。该研究将通过不同压力下的弛豫色散和化学交换饱和转移（CEST）实验来补充。

项目成果

Pressure dependence of side chain 1H and 15N-chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2

模型肽 Ac-Gly-Gly-Xxx-Ala-NH2 中侧链 1H 和 15N 化学位移的压力依赖性

• DOI: 10.1007/s10858-020-00326-w
• 发表时间: 2020
• 期刊: Journal of Biomolecular Nmr
• 影响因子: 2.7
• 作者: Beck Erlach; M.; Koehler; J.; Munte; C.E.; Kremer; W.; Crusca Jr; E.; Kainosho; M.; Kalbitzer; H. R.
• 通讯作者: H. R.

The pressure and temperature perturbation approach reveals a whole variety of conformational substates of amyloidogenic hIAPP monitored by 2D NMR spectroscopy.

压力和温度扰动方法揭示了通过 2D NMR 光谱监测的淀粉样变 hIAPP 的各种构象亚态

• DOI: 10.1016/j.bpc.2019.106239
• 发表时间: 2019
• 期刊: Biophysical chemistry
• 影响因子: 3.8
• 作者: Beck Erlach; M.; Kalbitzer; H. R.; Winter; R.; Kremer; W.
• 通讯作者: W.