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在构建基于亲和力和活性的全方位化蛋白质探针时,以铁为媒介的蛋白质全方位化

Gregory A Davidson¹, Zeinab Moafian¹, Amanda R Sensi¹

¹Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA.

Nature protocols

|April 25, 2025

概括

研究人员使用α-基策略开发了一种化学 ubiquitination 方法,以轻松创建单重和多重ubiquitinated 蛋白质. 这种多功能技术有助于研究蛋白质无处不在和识别蛋白质读取器和擦拭器.

科学领域:

生物化学生物化学
分子生物学分子生物学
后翻译修改后翻译修改

背景情况:

蛋白质泛基化是真核细胞中至关重要的调节机制,涉及泛基和蛋白之间的异键形成.
尽管具有重要意义,但为详细研究产生足够数量的同质单和多基化蛋白质仍然是一个重大挑战.
现有的酶和化学方法在可访问性和可扩展性方面存在限制,对于全面的无处不在化研究而言.

研究的目的:

介绍一种新的,易于化学 ubiquitination 方法,用于生成单基和多基 ubiquitinated 蛋白质.
为了能够创建具有特定修改的无处不在的蛋白质,例如可光激活的交叉链接器,用于捕获相互作用的蛋白质.
开发一个多功能平台,用于创建基于活动的探针,以研究duebiquitinases和ubiquitin ligases.

主要方法:

采用了α-基介导的结合策略,用于化学蛋白质无化.
引入了氨酸残留物到氨酸目标部位,并将本地氨酸突变为氨酸,以进行特定部位的修改.
展示了使用增殖细胞核抗原 (PCNA) 产生二-和三-均化PCNA的方法,使用可光激活的交叉链接器.

主要成果:

通过使用化学结合策略成功生成了单和多基化蛋白质.
开发了生产二-和三-无化PCNA的详细协议,以促进读者蛋白的捕获.
铁介导的结合和净化过程通常需要2-3周.
整合了迈克尔-接受器弹头,用于创建基于活动的探针,针对duebiquitinases和E3 ubiquitin ligases.

结论:

开发的化学ubiquitination方法为生成各种ubiquitinated蛋白质形式提供了一种多功能和可访问的方法.
这种方法显著提高了研究特定泛化事件的作用和识别蛋白质相互作用者的能力.
产生的基于活动的探测器为研究无处不在途径的动态和功能提供了强大的工具.

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