HIGH FREQUENCY EPR OF BIRADICAL SPIN LABELED PEPTIDES

定点定向自旋标记技术已经成为一种强大的 一种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技术用于 在双自由基体系中测量高达25A的自旋间距离 最近由Shin(Rabenstein，1995)和Beth(Hustedt， 1997年)。我们正在调查使用高频的优势。 EPR(HF-EPR)，用于提取距离和方向信息 双自由基自旋系统。我们正在研究螺旋肽的光谱 其中甲烷硫代磺酸盐(MTSSL)或TOAC(非天然的 以氮氧化物为侧链的氨基酸)的自旋标记 在两个选定的位点并入到肽序列中。这个 HF-EPR谱显示交换和 两个氮氧化物自旋标记物之间的偶极偶联。模拟 的实验数据正在进行中，以提取 双基距离分离和相对取向。初步 仿真结果表明，高频电子顺磁共振的光谱特征非常明显。 对两个自旋标签的相对方向敏感。这 使MTSSL双自由基肽的分析复杂化，其中 由于分子的移动性，获得了取向分布 系带氮氧化物自旋标签。相比之下，TOAC自转的刚性越强 标签更适合于方位信息的提取。

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.