The discovery and validation of protein-protein interactions provides a knowledge base that is critical for defining protein networks and how they underpin the biology of the cell. Identification of protein interactions that are highly transient, or sensitive to biochemical disruption, can be very difficult. This challenge has been met by proximity labeling methods which generate reactive species that chemically modify neighboring proteins. The most widely used proximity labeling method is BioID, which features a mutant biotin ligase BirA(Arg118Gly), termed BirA*, fused to a protein of interest. Here, we explore how amino acid substitutions at Arg118 affect the biochemical properties of BirA. We found that relative to wild-type BirA, the Arg118Lys substitution both slightly reduced biotin affinity and increased the release of reactive biotinyl-5'-AMP. BioID using a BirA(Arg118Lys)-Lamin A fusion enabled identification of PCNA as a lamina-proximal protein in HEK293T cells, a finding that was validated by immunofluorescence microscopy. Our data expand on the concept that proximity labeling by BirA fused to proteins of interest can be modulated by amino acid substitutions that affect biotin affinity and the release of biotinyl-5'-AMP.

中文翻译：

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通过生物素连接酶 BirA 改进邻近标记

蛋白质-蛋白质相互作用的发现和验证提供了一个知识库，这对于定义蛋白质网络以及它们如何支持细胞生物学至关重要。鉴定高度瞬态或对生化破坏敏感的蛋白质相互作用可能非常困难。这一挑战已通过邻近标记方法得到解决，该方法产生化学修饰邻近蛋白质的反应性物质。最广泛使用的邻近标记方法是 BioID，它具有突变的生物素连接酶 BirA(Arg118Gly)，称为 BirA*，与感兴趣的蛋白质融合。在这里，我们探讨了 Arg118 处的氨基酸取代如何影响 BirA 的生化特性。我们发现相对于野生型 BirA，Arg118Lys 取代既略微降低了生物素亲和力，又增加了活性生物素-5'-AMP 的释放。使用 BirA(Arg118Lys)-Lamin A 融合物的 BioID 能够将 PCNA 鉴定为 HEK293T 细胞中的近层蛋白，这一发现已通过免疫荧光显微镜验证。我们的数据扩展了一个概念，即通过与感兴趣的蛋白质融合的 BirA 进行邻近标记可以通过影响生物素亲和力和生物素-5'-AMP 释放的氨基酸取代进行调节。