New Nitroxide Spin Labels for Distance Measurements in Biological Systems

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 具体目标。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。

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.