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NMR光谱（HPNMR）代表一种强大的，仍在开发的方法来检测和表征稀有的“激发”构象状态。这些状态在蛋白质的功能和折叠中起着至关重要的作用。 HPNMR提供其热力学参数，但仅提供有限分辨率的结构信息。高压大分子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度分辨的空间结构。作为重要且特征良好的信号转导系统的组成部分，我们将集中在小的G蛋白Ras（大鼠肉瘤）蛋白上（发现Ras突变在所有人类癌症的30％以上都突变）。它是拟议项目中方法开发的特征良好模型，但新型的热力学和结构见解也来自很高的医学重要性，因为它们可能有助于发展变构RAS抑制剂的发展。通过与GTP模拟GPPNHP复合物的HPNMR，四个不同状态1（0），1（t），2（t）和3（t）可以在热力学上表征在环境压力下并存。优先结合状态1（t）的小型化合物可以变构抑制效应子的相互作用，因此原则上可以抑制致癌性RAS突变体的增殖作用。该项目的主要目标是（1）对H-和K-RAS的罕见构象状态及其与GDP和GPPNHP的罕见构象状态进行的替代NMR方法支持的替代NMR方法，例如CEST和放松分散测量，（2）可以提高概念的较高构度，从而有效地构成trap trap trap trap trap trap trap trap trap trap trap traps traps traps trap traps traps traps traps traps traps traps nmr。通过HPNMR在溶液中鉴定的NMR光谱法，（3）将观察到的NMR参数的压力响应与HPMX检测到的结构变化相关，并且（4）证明所获得的高压晶体学结构可用于设计特定的异源性抑制剂与稀有状态的结合。为了证明热力学上相似的状态SIN晶体和溶液在结构上也相似，从X射线结构中重新计算了种群加权的Noesy光谱，并与实验溶液状态noesy Spectra进行了比较。由于初步实验表明，具有小态特异性化合物的高压浸泡可能代表了通过晶体学进入罕见构象状态的新方法。在不同状态下的压力下的动力学将通过美国开发的高压跳跃系统上的2D瞬态P-跳跃实验进行研究。这项研究将通过在不同压力下的松弛分散体和化学交换饱和转移（CEST）实验来补充。