Crosslinking of ribosomal proteins by 4-(6-formyl-3-azidophenoxy)butyrimidate, a heterobifunctional, cleavable crosslinker

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Abstract
For the identification of neighbor relationships between proteins in biological systems, 4-(6-formyl-3-azidophenoxy)butyrimidate (FAPB-imidate), a heterobifunctional, cleavable cross-linker was synthesized. The reagent has an imido ester at one end, which is used for the attachment to amino groups of a specific protein whose environment has to be characterized. At the other end, the reagent has both an azido and an aldehyde group. The azido group can be used to cross-link the protein photochemically to a variety of chemical groups of neighboring proteins. The aldehyde group is able to cross-link the protein by reductive alkylation to amino groups of neighboring proteins. In both cases, the cross-linker can be made radioactive with NaB3H4. The cross-linked complexes can be split at the band originating from the imidate group by treatment with ammonia. The radioactive cross-linker remains covalently attached to the unknown neighboring protein, which can be therefore easily identified. FAPB-imidate was attached to [Escherichia coli] ribosomal protein L7. With this modified L7, the existence of the well-known complex between L7 and ribosomal protein L10 could be demonstrated by the photochemical procedure. By the chemical procedure, the presence of dimers of L7 in solution could be shown.