黄瓜[7]uril 增强了脊柱标记蛋白质的距离测量
Zhimin Yang1, Richard A Stein2, Maren Pink3
1Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, United States.
bioRxiv : the preprint server for biology
|September 4, 2023
概括
库库比特[7]uril (CB-7) 与一种新型的氧化物基 (ClA-DZD) 形成强烈的复合物,增强固定性,并使T4Lysozyme等生物分子中的敏感,远距离电子旋转测量成为可能.
科学领域:
- 超分子化学 超分子化学
- 生物物理化学 生物物理化学
- 电子偏磁共振光谱学 电子偏磁共振光谱学
背景情况:
- 有机基是探测分子动力学和结构的关键旋转标签.
- 黄瓜 (CBs) 是宏循环宿主,在分子识别和稳定方面具有潜在的应用.
- 之前的研究已经探索了激素与宿主相互作用,但对于先进的EPR应用来说,高 afinity系统是有限的.
研究的目的:
- 为了研究一种乙 2,6-亚扎亚达曼坦氧化基 (ClA-DZD) 和甲[7]uril (CB-7) 之间的复杂形成.
- 评估CB-7封装对基因动态的影响及其在电子磁共振 (EPR) 测量中的实用性,特别是双电子共振 (DEER).
- 探索CB-7复合的应用,以增强对自旋标记蛋白质的DEER测量.
主要方法:
- 在水溶液中确定复杂的形成和结合常量.
- 使用EPR的旋转相关时间 (τ_rot) 测量.
- 进行X射线晶体学以确定ClA-DZD@CB-7复合体的结构.
- 在存在或缺少CB-7的情况下,对自旋标记的T4溶酶 (T4L) 进行DEER光谱.
主要成果:
- 对ClA-DZD@CB-7复合物的高关联常数 (Ka = 1.9 × 10^6 M^-1),明显高于之前研究的有机基.
- CB-7强烈地固定了CLA-DZD,增加了t_rot的36倍.
- X射线结构显示,CLA-DZD被封装在CB-7内,悬挂组暴露在外.
- CB-7添加到旋转标记的T4L增强了DEER的灵敏度 (4-9倍),并允许可靠地测量3nm间旋转距离,最高可达200K.
- DZD-T4L的X射线结构显示了与DZD标签无干扰的蛋白质构成.
结论:
- CB-7与ClA-DZD基形成了一个高度稳定的复合体,显著改变了它的动态.
- CB-7复合增强了对自旋标记蛋白的DEER测量的灵敏度和温度范围.
- 这种超分子方法为近乎生理条件下的远距离EPR测量提供了一个有希望的策略.
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