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在活细胞中通过控制编码的素的氧化状态来定量标记蛋白质

Alex J Eddins¹, Yogesh M Gangarde¹, Anamika Singh¹

¹Department of Biochemistry and Biophysics & GCE4All Research Center, Oregon State University, 2011 Agricultural and Life Sciences, Corvallis, Oregon 97331, United States.

Journal of the American Chemical Society

|June 25, 2025

概括

在活细胞中通过编码1,2,4,5-四素 (Tet) 残留物来进行特定位点的蛋白质标记. 新方法可以控制TET氧化还原状态,通过光激活进行快速和完整的蛋白质标记.

科学领域:

化学生物学
生物技术
分子成像

背景情况:

在生物研究和药物开发中,特定区域的蛋白质修饰至关重要.
1,2,4,5-四 (Tet) 残留物为生物系统的定量标记提供了快速的动力学.
细胞内氧化还原环境可能会影响Tet的反应性,限制标记效率.

研究的目的:

开发和验证在活细胞中评估和改进基于Tet的蛋白质标签的方法.
研究细胞内氧化还原状态对TET残留物反应性的影响.
设计具有可调节的氧化还原潜力的新型TET变体,

主要方法:

将凝转移试验改为"PEG Chaser试验",以评估标签的完整性.
使用光氧化来将减少的TET形式转化为反应性氧化形式.
使用遗传代码扩展来编码具有不同氧化还原潜力的新型TET非正规氨基酸 (ncAAs).

主要成果:

在活细胞中,PEG Chaser检测成功地区分了标记和未标记的蛋白质.
在氧化 (Tz) 和减少 (DHTz) 形式之间的平衡中存在编码的 Tet 残留物.
新的 Tet3H ncAA 能够在 5 分钟内进行完全蛋白质修饰的光激活标记.

结论:

控制 Tet 残留物的氧化还原状态是优化细胞内标签的关键策略.
具有可调节的氧化还原潜力的工程 Tet ncAAs显著扩大了 Tet 化学在活细胞中的实用性.
可光激活的TET标签为生物系统中的定量蛋白质修饰提供了一种快速有效的方法.

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