脉冲ESR双极光谱用于液体溶液中固定自旋标记蛋白质的距离测量
Zhongyu Yang1, Yangping Liu, Peter Borbat
1Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Journal of the American Chemical Society
|June 9, 2012
概括
研究人员开发了一种使用三甲基 (TAM) 旋转标签用于脉冲电子旋转共振 (ESR) 二极光谱 (PDS) 的新方法. 这种技术允许在生物系统中测量液体溶液中的距离,而不仅仅是冷状态.
科学领域:
- 生物物理学的生物物理.
- 结构生物学 结构生物学
- 频谱学是一种光谱学.
背景情况:
- 脉冲电子自旋共振 (ESR) 双极光谱 (PDS) 与位点定向的自旋标记测量生物系统中的纳米距离.
- 传统的PDS需要冷温度,由于快速的电子自旋放松和分子翻转,限制了液体溶液中的测量.
研究的目的:
- 为了使纳米范围的距离测量使用PDS在液体溶液中.
- 探索基于三甲基 (TAM) 的旋转标签的实用性,对于固定蛋白质具有很长的放松时间.
主要方法:
- 通过二硫化物链接将T4溶酶固定在Sepharose上.
- 在固定的T4溶酶上,将TAM基与特定的氨酸残留物 (65/80或65/76) 结合在一起.
- 使用双量子连贯性 (DQC) 来确定液体溶液中的间旋距离.
主要成果:
- 在溶液中确定的互旋距离与模型预测密切匹配.
- 观察到的窄距离分布表明本地化了基于TAM的旋转标签.
- 通过在固定蛋白质上使用TAM标签在液体溶液中进行PDS测量的可行性.
结论:
- 基于TAM的旋转标签为液体溶液中的PDS测量提供了一种可行的方法.
- 蛋白质在坚实的支上固定,克服了快速分子翻转的局限性.
- 这种方法扩大了PDS在接近本地条件下研究生物系统的适用性.
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