Protein-Protein Interface Identification by Site-Specific Photo-Cross-linking/Cleavage in Mammalian Cells.

Autor: Terasawa K; Department of Biochemistry, Tokyo Medical and Dental University (TMDU), Bunkyo-ku, Tokyo, Japan.; LiberoThera Co., Ltd., Chuo-ku, Tokyo, Japan., Seike T; Department of Periodontology, Tokyo Medical and Dental University (TMDU), Bunkyo-ku, Tokyo, Japan., Sakamoto K; Laboratory for Nonnatural Amino Acid Technology, RIKEN Center for Biosystems Dynamics Research, RIKEN, Tsurumi-ku, Yokohama, Japan.; Department of Drug Target Protein Research, Shinshu University School of Medicine, Matsumoto, Nagano, Japan., Ohtake K; Laboratory for Nonnatural Amino Acid Technology, RIKEN Center for Biosystems Dynamics Research, RIKEN, Tsurumi-ku, Yokohama, Japan.; Department of Electrical Engineering and Bioscience, Waseda University, Shinjuku, Tokyo, Japan., Watabe T; Department of Biochemistry, Tokyo Medical and Dental University (TMDU), Bunkyo-ku, Tokyo, Japan.; Department of Structural Biology and Biochemistry, Tokyo Medical and Dental University (TMDU), Bunkyo-ku, Tokyo, Japan., Yokoyama S; Department of Drug Target Protein Research, Shinshu University School of Medicine, Matsumoto, Nagano, Japan.; Department of Structural Biology and Biochemistry, Tokyo Medical and Dental University (TMDU), Bunkyo-ku, Tokyo, Japan.; Laboratory for Protein Function and Structural Biology, RIKEN Cluster for Science, RIKEN, Tsurumi-ku, Yokohama, Japan., Hara-Yokoyama M; Department of Biochemistry, Tokyo Medical and Dental University (TMDU), Bunkyo-ku, Tokyo, Japan.
Jazyk: angličtina
Zdroj: Current protocols [Curr Protoc] 2024 Aug; Vol. 4 (8), pp. e1103.
DOI: 10.1002/cpz1.1103
Abstrakt: Identification of protein-protein interfaces is necessary for understanding and regulating biological events. Genetic code expansion enables site-specific photo-cross-linking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the cross-linked region still remains challenging. Our new protocol enables its identification by pre-installing a site-specific cleavage site, an α-hydroxy acid (N ε -allyloxycarbonyl-α-hydroxyl-L-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located on within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. This combination of site-specific crosslinking and cleavage promises to be useful for revealing binding interfaces and protein complex geometries. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Search for crosslinkable sites Basic Protocol 2: Site-specific photo-cross-linking/cleavage.
(© 2024 Wiley Periodicals LLC.)
Databáze: MEDLINE
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