HIGH FREQUENCY EPR OF BIRADICAL SPIN LABELED PEPTIDES

双自由基自旋标记肽的高频 EPR

• 批准号: 6355139
• 负责人: CHRISTIAN T FARRAR
• 金额: $ 2.14万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6355139
• 关键词: biological products; biomedical resource; drug /agent; nucleic acids

The technique of site-directed spin labeling has become a powerful tool for studying protein secondary structure and protein dynamics by EPR spectroscopy. In addition, low frequency CW-EPR techniques for measuring interspin distances of up to 25 A in biradical systems have recently been developed by Shin (Rabenstein, 1995) and Beth (Hustedt, 1997). We are investigating the advantages of using high frequency EPR (HF-EPR) to extract both distance and orientation information from biradical spin systems. We are examining spectra of helical peptides where either the methanthiosulfonate (MTSSL) or TOAC (an unnatural amino acid with a nitroxide as the side chain) spin labels have been incorporated into the peptide sequence at two selected sites. The HF-EPR spectra demonstrate splittings arising from exchange and dipolar couplings between the two nitroxide spin labels. Simulations of the experimental data are currently underway to extract the biradical distance separations and relative orientations. Preliminary simulations indicate that the HF-EPR spectral features are very sensitive to the relative orientation of the two spin labels. This complicates the analysis of the MTSSL biradical peptides where a distribution of orientations is obtained due to the mobility of the tethered nitroxide spin label. In contrast, the more rigid TOAC spin label is more amenable to the extraction of orientation information.

定点自旋标记技术已经成为一种强有力的 研究蛋白质二级结构和蛋白质动力学的工具， 电子顺磁共振波谱。 此外，低频CW-EPR技术 在双自由基系统中测量高达25 A的自旋间距离， 最近由Shin（Rabenstein，1995）和Beth（胡斯特， 1997年）。 我们正在研究使用高频的优点 EPR（HF-EPR），以从 双自由基自旋系统 我们正在研究螺旋肽的光谱 其中甲硫基磺酸盐（MTSSL）或TOAC（非天然的 具有氮氧自由基作为侧链的氨基酸）自旋标记物已经被 在两个选择的位点掺入肽序列。 的 HF-EPR谱表明分裂产生的交换和 两个氮氧自旋标记之间的偶极耦合。 模拟 的实验数据目前正在进行中，以提取 双基距离分离和相对取向。 初步 模拟结果表明，HF-EPR谱特征非常 对两个自旋标记的相对取向敏感。 这 使MTSSL双自由基肽的分析复杂化， 取向的分布是由于获得的流动性， 系留氮氧自旋标记。 相比之下，更刚性的TOAC自旋 标签更易于提取方向信息。