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。

基本信息

项目摘要

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)代表了一种强大的,仍在发展的方法来检测和表征罕见的“激发态”构象状态。这些状态在蛋白质的功能和折叠中起着重要的作用。HPNMR提供了它们的热力学参数,但只给出了有限分辨率的结构信息。高压大分子x射线晶体学(HPMX)是蛋白质科学中一种互补的新方法,基本上可以在高压下提供高分辨率的空间结构。作为一个重要且特征明确的信号转导系统的组成部分,我们将集中研究小g蛋白Ras(大鼠肉瘤)蛋白(Ras突变在超过30%的人类癌症中被发现发生突变)。它可以作为拟议项目中方法开发的良好表征模型,但新的热力学和结构见解也具有很高的医学重要性,因为它们可能有助于开发变构性Ras抑制剂。通过与GTP类似物GppNHp配合物的HPNMR,可以表征出在环境压力下共存的4种状态1(0)、1(T)、2(T)和3(T)。优先结合状态1(T)的小化合物可以变变地抑制效应物相互作用,因此原则上可以抑制致癌Ras突变体的增殖作用。本项目的主要目标是:(1)在CEST和弛驰色散测量等替代核磁共振方法的支持下,通过HPNMR对H-和K-Ras及其致癌突变体与GDP和GppNHp复合物中罕见的构象状态进行彻底的定量分析;(2)证明HPMX可以有效地捕获高压核磁共振光谱在溶液中鉴定的蛋白质的高能构象。(3)将观察到的核磁共振参数的压力响应与HPMX检测到的结构变化联系起来;(4)原则上,得到的高压晶体结构可以用于设计特异性的与稀有态结合的变构抑制剂。为了证明晶体和溶液的热力学相似态是否在结构上也相似,根据x射线结构反演了种群加权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,用小态特定化合物高压浸泡可能是一种通过晶体学获得稀有构象态的新方法。利用自制的高压跳变系统,对富同位素Ras蛋白进行二维瞬态p跳实验,研究其在不同状态下的动力学特性。本研究将辅以不同压力下的弛豫分散和化学交换饱和转移(CEST)实验。

项目成果

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Professor Dr. Hans Robert Kalbitzer其他文献

Professor Dr. Hans Robert Kalbitzer的其他文献

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{{ truncateString('Professor Dr. Hans Robert Kalbitzer', 18)}}的其他基金

Characterization of different conformational states of proteins by highpressure NMR spectroscopy
通过高压核磁共振波谱表征蛋白质的不同构象状态
  • 批准号:
    243247384
  • 财政年份:
    2013
  • 资助金额:
    --
  • 项目类别:
    Research Units
3D-Struktur des Cannulae bildenden Proteins CanA des hyperthermophilen Archaeum Pyrodictium spec. in atomarer Auflösung
超嗜热古细菌 Pyrodictium 种的插管形成蛋白 CanA 的 3D 结构。
  • 批准号:
    195109522
  • 财政年份:
    2011
  • 资助金额:
    --
  • 项目类别:
    Research Grants
NMR-Struktur von Thioredoxin und Plasmoredoxin von Plasmodium falciparum und Charakterisierung seiner Komplexe mit interagierernden Proteinen
恶性疟原虫硫氧还蛋白和胞质氧还蛋白的 NMR 结构及其与相互作用蛋白的复合物的表征
  • 批准号:
    35121158
  • 财政年份:
    2006
  • 资助金额:
    --
  • 项目类别:
    Research Grants
Struktur und Dynamik des Protoonkogens Ras und spezifischer Ras-Mutanten.
原癌基因 Ras 和特定 Ras 突变体的结构和动力学。
  • 批准号:
    21540907
  • 财政年份:
    2006
  • 资助金额:
    --
  • 项目类别:
    Research Grants
Entwicklung einer spektrometergesteuerten Druckapparatur zur Hochdruck-NMR-Spektroskopie für die Untersuchung von konformationellen Intermediaten
开发用于高压核磁共振波谱研究的波谱仪控制压力装置,用于研究构象中间体
  • 批准号:
    17772873
  • 财政年份:
    2005
  • 资助金额:
    --
  • 项目类别:
    Research Grants
Lösungsstruktur der Ras-Bindungsdomäne von Nore1 und strukturelle Charakterisierung der Nore1-Ras-Mst1-Interaktion
Nore1 Ras 结合域的溶液结构以及 Nore1-Ras-Mst1 相互作用的结构表征
  • 批准号:
    5404508
  • 财政年份:
    2003
  • 资助金额:
    --
  • 项目类别:
    Research Grants
Solution structure of the Ras-binding domain of AF6 and its interaction with the Ras-protein
AF6 Ras 结合域的溶液结构及其与 Ras 蛋白的相互作用
  • 批准号:
    5252292
  • 财政年份:
    2000
  • 资助金额:
    --
  • 项目类别:
    Research Grants
Struktur und Funktion viraler Proteine: Nef-Protein von HIV-2
病毒蛋白的结构和功能:HIV-2的Nef蛋白
  • 批准号:
    5168418
  • 财政年份:
    1999
  • 资助金额:
    --
  • 项目类别:
    Research Units
High-Field EPR/ENDOR Spectroscopy on Guanine Nucleotide Binding Protein
鸟嘌呤核苷酸结合蛋白的高场 EPR/ENDOR 光谱
  • 批准号:
    5131932
  • 财政年份:
    1998
  • 资助金额:
    --
  • 项目类别:
    Priority Programmes
Lösungsstruktur der Ran-Bindungsdomäne von RanBP2 und Protein-Protein-Interaktionenen im Ran-gekoppelten Kerntransportsystem
RanBP2 Ran结合域的溶液结构和Ran耦合核转运系统中蛋白质-蛋白质相互作用
  • 批准号:
    5077246
  • 财政年份:
    1997
  • 资助金额:
    --
  • 项目类别:
    Priority Programmes

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Rare Metals(稀有金属(英文版))
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