A Method for Protein Photo-cross-linking in Living Cells Facilitating Analysis of Physiological Interactions of Proteins

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  • Hino Nobumasa
    Graduate School of Pharmaceutical Sciences, Osaka University

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  • 生理的なタンパク質間相互作用の解析を可能にする細胞内光クロスリンク技術
  • セイリテキ ナ タンパクシツ カン ソウゴ サヨウ ノ カイセキ オ カノウ ニ スル サイボウ ナイコウ クロスリンク ギジュツ

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Abstract

  In living cells, most proteins form complexes with other proteins to exert their functions. Since protein functions are regulated in response to changes in the cellular environment, the components of the complexes can vary; therefore, proteins often interact in a weak and transient manner. To capture such labile protein interactions, we have developed a method for photo-cross-linking of proteins directly interacting in mammalian cells; this method involves expansion of the genetic code and site-specific incorporation of photoreactive amino acids into proteins. Upon cross-linking, protein complexes are stabilized by a covalent bond and can be readily isolated from cell extracts without the problems usually associated with simple affinity purification methods such as co-immunoprecipitation. Photo-cross-linkers have another benefit: they react exclusively with molecules within a range defined by the linker length. This property becomes useful for determining the binding interface of two proteins because the linkers can be introduced in a site-directed manner with our method. In this review, we first describe the expansion of the genetic code of mammalian cells for the incorporation of non-natural amino acids into proteins. Then, we introduce our recent applications and developments of the cross-linking method: identification of intracellular binding partners of the signaling protein growth factor receptor binding protein 2; analysis of the binding between membrane proteins on the cell surface; and a novel photoreactive amino acid that enables wide-ranging photo-cross-linking.<br>

Journal

  • YAKUGAKU ZASSHI

    YAKUGAKU ZASSHI 135 (12), 1357-1363, 2015-12-01

    The Pharmaceutical Society of Japan

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