title: A class of cleavable heterobifunctional reagents for thiol-directed high-efficiency protein crosslinking: synthesis and application to the analysis of protein contact sites in mammalian ribosomes.
abstract: The synthesis of a new class of cleavable crosslinking reagents is described. The primary function, a ring-substituted maleimide, binds selectively and very efficiently at low pH to cysteine-containing protein sequences. At increased pH the secondary function, an N-hydroxysuccinimide ester of a ring-attached carboxyl group, becomes reactive against adjacent amino groups. The spacer, azobenzene, is readily cleaved by reduction with dithionite provided that a hydroxyl group is included in the ring system. By altering the relative positions of the reactive groups the range of crosslinking can be varied within approximately 8-12 A. After degradation of the crosslinked proteins by optional methods the contact sequences are readily identified by diagonal electrophoresis. By radiolabeling the carboxyl group of the reagent the order of binding of the proteins can be established, and the secondarily attached protein and its contact sequences can be recognized even in trace amounts. The usefulness of the reagents is illustrated by the selective, high-efficiency crosslinking of mammalian ribosomal proteins and the identification of their contact fragments as obtained by CNBr degradation.
mesh_terms: D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D011485:Protein Binding; D012269:Ribosomal Proteins / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000032:analysis*
chemical_list: D003432:Cross-Linking Reagents; D012269:Ribosomal Proteins; D013438:Sulfhydryl Compounds; D003488:Cyanogen Bromide