New Nitroxide Spin Labels for Distance Measurements in Biological Systems

用于生物系统距离测量的新型氮氧化物自旋标签

基本信息

  • 批准号:
    9922926
  • 负责人:
  • 金额:
    $ 41.29万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2017
  • 资助国家:
    美国
  • 起止时间:
    2017-08-01 至 2023-04-30
  • 项目状态:
    已结题

项目摘要

Project Summary Nitroxide spin labels are useful probes for investigation of biomacromolecules such as proteins DNA, and RNA. In a typical approach, doubly-labeled proteins are prepared by site-directed spin labeling (SDSL) and a set of distances between spin labels is measured using pulsed electron paramagnetic resonance (EPR) dipolar spectroscopy (PDS). Double electron electron resonance (DEER) is the most widely used PDS method. The temperatures at which the DEER measurements currently can be made are limited by the dynamic averaging effects associated with methyl group rotation in the typical nitroxide spin label, and therefore have to be performed at about 50 – 70 K, requiring use of liquid helium, or with lower sensitivity at 80 K with liquid nitrogen. In addition, application of the SDSL methodology to in vivo measurements is hampered by the short in vivo lifetime of the currently available nitroxide spin labels. The objective of the proposed work is to synthesize and evaluate new nitroxide spin labels for DEER distance measurements in proteins at physiological temperature and for in vivo DEER at cryogenic temperature. The proposed project has three specific aims. Aim 1: synthesize ultra-rigid, small-sized, and polar nitroxide spin labels for distance measurements at physiological temperatures (D-Labels). The target labels are novel nitroxides devoid of methyl groups with structure motifs that address the problem of conformational flexibility, molecular size and hydrophobicity. Aim 2: synthesize nitroxide spin labels for in vivo DEER distance measurements (C-Labels). The targets labels are biostable gem-diethyl and gem-dicarboxylate nitroxide spin labels. Aim 3: evaluate effectiveness of the synthesized spin labels for SDSL-DEER distance measurements at physiological temperatures and for in vivo DEER distance measurements. All labels synthesized in Aims 1 and 2 will be tested for their electron spin relaxation times (Tm and T1) as a function of temperature up to 310 K and for the rate of reduction in ascorbate/glutathione solutions. SDSL-DEER distance measurements at physiological temperature will be calibrated using T4 Lysozyme (T4L) that is doubly spin labelled with D-Labels and immobilized in carbohydrate matrix. C-Labels will be tested for in vivo DEER distance measurements at cryogenic temperature, via SDSL of selected proteins in live mitochondria and in E. coli. Collaborating laboratories will carry out these studies and set the stage for the general adoption of the proposed spin labels by highlighting their properties and defining the range of their applications. These studies will provide structural information at physiological temperature and/or in the native cellular environment, which is characterized by factors and conditions that may have a crucial role in determining the biologically relevant conformation of the proteins under investigation. The wealth of information enabled by the proposed labels will be close to ideal for understanding diseases and the approaches to developing cures for diseases.
项目摘要 氮氧自由基自旋标记物是研究蛋白质、DNA、RNA等生物大分子的有效探针。 在一种典型的方法中,通过定点自旋标记(SDSL)和一组双标记蛋白来制备双标记蛋白。 使用脉冲电子顺磁共振(EPR)偶极测量自旋标记之间的距离 光谱学(PDS)。双电子电子共振(DEER)是应用最广泛的PDS方法。的 目前可以进行DEER测量的温度受到动态平均的限制 在典型的氮氧自旋标记中与甲基旋转相关的效应,因此必须 在大约50 - 70 K下进行,需要使用液氦,或者在80 K下用液体 氮气此外,SDSL方法在体内测量中的应用受到短时间的限制。 目前可用的氮氧自旋标记物的体内寿命。拟议工作的目标是 合成和评价用于蛋白质中DEER距离测量新氮氧自旋标记物. 生理温度和低温下的体内DEER。该项目有三个 明确的目标。目标1:合成超刚性,小尺寸,极性氮氧自由基自旋标记物的距离 生理温度下的测量(D标签)。靶标记物是不含以下的新型氮氧自由基: 具有解决构象灵活性、分子大小和 疏水性目的2:合成用于体内DEER距离测量的氮氧自由基自旋标记物(C-Labels)。 目标标记物是生物稳定的偕-二乙基和偕-二羧酸氮氧自由基自旋标记物。目标3:评价 合成自旋标记在生理条件下用于SDSL-DEER距离测量的有效性 温度和体内DEER距离测量。在目标1和2中合成的所有标记物将 测试了它们的电子自旋弛豫时间(Tm和T1)作为温度的函数,最高可达310 K, 抗坏血酸/谷胱甘肽溶液中的还原速率。SDSL-DEER距离测量在生理 将使用T4溶菌酶(T4 L)校准温度,T4溶菌酶(T4 L)用D-标记进行双重自旋标记, 固定在碳水化合物基质中。将测试C-标签的体内DEER距离测量值, 低温下,通过SDSL的选择蛋白质在活线粒体和在E.杆菌合作 实验室将进行这些研究,并为普遍采用拟议的自旋标签奠定基础 通过突出它们的特性和定义它们的应用范围。这些研究将提供结构 在生理温度下和/或在天然细胞环境中的信息,其特征在于 这些因素和条件可能在确定生物学相关构象中起关键作用, 正在研究的蛋白质拟议的标签所提供的丰富信息将接近理想, 了解疾病和开发疾病治疗方法的方法。

项目成果

期刊论文数量(11)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels
使用 DEER 和生物抗性正交自旋标签对膜嵌入 Bax 寡聚物的新视角
  • DOI:
    10.1038/s41598-019-49370-z
  • 发表时间:
    2019
  • 期刊:
  • 影响因子:
    4.6
  • 作者:
    Teucher;Winklhofer;García-Sáez;Bleicken;Bordignon
  • 通讯作者:
    Bordignon
Bis-Spiro-Oxetane and Bis-Spiro-Tetrahydrofuran Pyrroline Nitroxide Radicals: Synthesis and Electron Spin Relaxation Studies
双螺环氧杂环丁烷和双螺四氢呋喃吡咯啉氮氧化物自由基:合成和电子自旋弛豫研究
  • DOI:
    10.1021/acs.joc.1c01670
  • 发表时间:
    2021
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Huang, Shengdian;Pink, Maren;Ngendahimana, Thacien;Rajca, Suchada;Eaton, Gareth R.;Eaton, Sandra S.;Rajca, Andrzej
  • 通讯作者:
    Rajca, Andrzej
High-Spin Diradical Dication of Chiral π-Conjugated Double Helical Molecule
Thermally and Magnetically Robust Triplet Ground State Diradical
Thermally Ultrarobust S = 1/2 Tetrazolinyl Radicals: Synthesis, Electronic Structure, Magnetism, and Nanoneedle Assemblies on Silicon Surface
耐热超鲁棒 S = 1/2 四唑啉基自由基:硅表面上的合成、电子结构、磁性和纳米针组件
  • DOI:
    10.1021/jacs.3c03402
  • 发表时间:
    2023
  • 期刊:
  • 影响因子:
    15
  • 作者:
    Yang, Zhimin;Pink, Maren;Nowik-Boltyk, Ewa Malgorzata;Lu, Shutian;Junghoefer, Tobias;Rajca, Suchada;Stoll, Stefan;Casu, Maria Benedetta;Rajca, Andrzej
  • 通讯作者:
    Rajca, Andrzej
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ANDRZEJ RAJCA其他文献

ANDRZEJ RAJCA的其他文献

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{{ truncateString('ANDRZEJ RAJCA', 18)}}的其他基金

New Nitroxide Spin Labels for Distance Measurements in Biological Systems
用于生物系统距离测量的新型氮氧化物自旋标签
  • 批准号:
    9364760
  • 财政年份:
    2017
  • 资助金额:
    $ 41.29万
  • 项目类别:
Synthesis of Metal-Free Magnetic Resonance Imaging Contrast Agents
无金属磁共振成像造影剂的合成
  • 批准号:
    9317484
  • 财政年份:
    2015
  • 资助金额:
    $ 41.29万
  • 项目类别:
High-Spin Nitroxide Radical for Biomedical Imaging Applications
用于生物医学成像应用的高自旋氮氧自由基
  • 批准号:
    7686790
  • 财政年份:
    2008
  • 资助金额:
    $ 41.29万
  • 项目类别:
High-Spin Nitroxide Radical for Biomedical Imaging Applications
用于生物医学成像应用的高自旋氮氧自由基
  • 批准号:
    7588405
  • 财政年份:
    2008
  • 资助金额:
    $ 41.29万
  • 项目类别:

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