Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling.
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.
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine.
Sarah A
SA
Head
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine; Department of Oncology, Johns Hopkins University School of Medicine; joliu@jhu.edu.
Jun O
JO
Liu
D002352:Carrier Proteins; D006801:Humans; D008565:Membrane Proteins; D019920:Photoaffinity Labels; D010777:Photochemistry
D002352:Carrier Proteins; D008565:Membrane Proteins; D019920:Photoaffinity Labels
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