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Genetically encoded releasable photo-cross-linking strategies for studying protein-protein interactions in living cells.

Authors
  • Yang, Yi1
  • Song, Haiping1
  • He, Dan1
  • Zhang, Shuai1
  • Dai, Shizhong1
  • Xie, Xiao1
  • Lin, Shixian1
  • Hao, Ziyang1
  • Zheng, Huangtao2
  • Chen, Peng R1, 2
  • 1 Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China. , (China)
  • 2 Peking-Tsinghua Center for Life Sciences, Beijing, China. , (China)
Type
Published Article
Journal
Nature protocols
Publication Date
Oct 01, 2017
Volume
12
Issue
10
Pages
2147–2168
Identifiers
DOI: 10.1038/nprot.2017.090
PMID: 28933779
Source
Medline
License
Unknown

Abstract

Although protein-protein interactions (PPIs) have crucial roles in virtually all cellular processes, the identification of more transient interactions in their biological context remains challenging. Conventional photo-cross-linking strategies can be used to identify transient interactions, but these approaches often suffer from high background due to the cross-linked bait proteins. To solve the problem, we have developed membrane-permeable releasable photo-cross-linkers that allow for prey-bait separation after protein complex isolation and can be installed in proteins of interest (POIs) as unnatural amino acids. Here we describe the procedures for using two releasable photo-cross-linkers, DiZSeK and DiZHSeC, in both living Escherichia coli and mammalian cells. A cleavage after protein photo-cross-linking (CAPP ) strategy based on the photo-cross-linker DiZSeK is described, in which the prey protein pool is released from a POI after affinity purification. Prey proteins are analyzed using mass spectrometry or 2D gel electrophoresis for global comparison of interactomes from different experimental conditions. An in situ cleavage and mass spectrometry (MS)-label transfer after protein photo-cross-linking (IMAPP) strategy based on the photo-cross-linker DiZHSeC is also described. This strategy can be used for the identification of cross-linking sites to allow detailed characterization of PPI interfaces. The procedures for photo-cross-linker incorporation, photo-cross-linking of interaction partners and affinity purification of cross-linked complexes are similar for the two photo-cross-linkers. The final section of the protocol describes prey-bait separation (for CAPP) and MS-label transfer and identification (for IMAPP). After plasmid construction, the CAPP and IMAPP strategies can be completed within 6 and 7 d, respectively.

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