New Nitroxide Spin Labels for Distance Measurements in Biological Systems

项目摘要 氮氧化物自旋标记是研究蛋白质、DNA和RNA等生物大分子的有用探针。 在一种典型的方法中，双标记蛋白质是通过定点定向自旋标记(SDSL)和一组 使用脉冲电子顺磁共振(EPR)偶极来测量自旋标记物之间的距离 光谱学(PDS)双电子共振(DER)是目前应用最广泛的PDS方法。这个 目前可以进行鹿测量的温度受到动态平均的限制 在典型的氮氧化物自旋标记中与甲基旋转相关的影响，因此必须 在约50-70K下进行，需要使用液氦，或在80K下使用液体时灵敏度较低 氮气。此外，SDSL方法在活体测量中的应用受到短 目前可用的氮氧化物自旋标记的活体寿命。拟议工作的目标是 合成和评价用于测量蛋白质中鹿距离的新的氮氧自旋标记 生理温度和体内鹿在低温下。拟议的项目有三个 明确的目标。目标1：合成超刚性、小尺寸和极性氮氧化物自旋标记物 生理温度下的测量(D-标签)。目标标记物是新型氮氧化物，不含 具有结构基序的甲基，解决构象灵活性、分子大小和 疏水性。目的2：合成用于体内鹿距离测量的氮氧化物自旋标记物(C-标记物)。 目标标记物是生物稳定的GEM-二乙基和GEM-二羧酸盐氮氧化物自旋标记物。目标3：评估 人工合成的自旋标记物在生理条件下测量SDSL-鹿距离的有效性 用于体温和活体鹿距离测量。在AIMS 1和2中合成的所有标签将 测试了它们的电子自旋弛豫时间(Tm和t1)在310K以下随温度的变化以及 抗坏血酸/谷胱甘肽溶液的减少速率。SDSL-生理学条件下的鹿距离测量 温度将使用T4溶菌酶(T4L)进行校准，T4溶菌酶(T4L)是用D-标记和 固定在碳水化合物基质中。C标记将在活体内测试鹿的距离测量 低温温度，通过在活的线粒体和大肠杆菌中选择的蛋白质的SDSL。协作 实验室将进行这些研究，并为拟议的自旋标签的普遍采用奠定基础 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上的 生理温度和/或自然蜂窝环境中的信息，其特征是 可能在确定与生物相关的构象中起关键作用的因素和条件 正在研究的蛋白质。建议的标签提供的丰富信息将接近理想的 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.