Identification of small molecule-binding proteins in a native cellular environment by live-cell photoaffinity labeling

Sarah A. Head, Jun Liu

Research output: Contribution to journalArticle

Abstract

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

Original languageEnglish (US)
Article numbere54529
JournalJournal of Visualized Experiments
Volume2016
Issue number115
DOIs
StatePublished - Sep 20 2016

Fingerprint

Labeling
Carrier Proteins
Molecules
Drug Discovery
Cell Communication
Membrane Proteins
Proteins
Maintenance
Membranes

Keywords

  • Biochemistry
  • Biochemistry
  • Chemical biology
  • Click chemistry
  • Drug discovery
  • Issue 115
  • Pharmacology
  • Photoaffinity
  • Target identification

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Identification of small molecule-binding proteins in a native cellular environment by live-cell photoaffinity labeling. / Head, Sarah A.; Liu, Jun.

In: Journal of Visualized Experiments, Vol. 2016, No. 115, e54529, 20.09.2016.

Research output: Contribution to journalArticle

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