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在人体细胞中直接表达化蛋白

Lan B T Pham¹, Azzurra Costantino¹, Letizia Barbieri^1,2

¹CERM─Magnetic Resonance Center, Università degli Studi di Firenze, Via Luigi Sacconi 6, 50019Sesto Fiorentino, Italy.

Journal of the American Chemical Society

|January 5, 2023

概括

-19 (F) 核磁共振可通过克服信号损失问题来研究人体细胞内的蛋白质行为. 这种方法很容易结合化氨基酸,使以前无法检测到的蛋白质及其相互作用可视化.

科学领域:

生物物理
结构生物学
细胞生物学

背景情况:

细胞内核磁共振光谱对原生细胞环境中的蛋白质结构和功能提供了洞察力.
传统的NMR方法面临诸如因蛋白与细胞组件相互作用而导致的线条扩展等挑战.
通过检测蛋白质中的原子,使得无背景光谱成为一个解决方案.

研究的目的:

开发一种简单的方法将化氨基酸纳入人体细胞表达的蛋白质中.
为了证明F细胞内NMR对传统NMR不适应的蛋白质的有用性.
在细胞环境中研究蛋白质 - 连接体相互作用和蛋白质 - 沙佩龙复合体动态.

主要方法:

为了在人类细胞中的蛋白质表达过程中有效地纳入化氨基酸,采用了中等切换策略.
使用细胞内NMR光谱分析化蛋白质.
使用质谱和X射线结晶学来确认化效率和结构.

主要成果:

在人体细胞中表达的蛋白质中达到高化效率 (高达60%).
成功地对各种蛋白质进行F细胞内核磁共振,包括对H-N核磁共振看不见的蛋白质.
观察了碳酸无水酶2及其抑制剂之间的相互作用,并研究了CCS与SOD1结合的调节.

结论:

介质切换策略为人类细胞中的蛋白质提供了一种多功能和高效的方法.
细胞内NMR是研究蛋白质动力学和它们在本土环境中的相互作用的强大工具.
这种方法扩大了细胞内NMR的范围,用于研究具有挑战性的生物系统和药物点.

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