Crosslinking of Ribosomes by Cleavable Bifunctional Mercaptoimidates

  • Robert R. Traut
  • James W. Kenny
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 86A)


Methods are described for determining protein:protein proximity relationships in complex cellular structures containing multiple protein components, Studies on the 50S ribosomal subunit of Escherichia coli illustrate the procedures employed. The intact ribosomal subunit is first incubated with methyl 4-mercaptobutyrimidate. Lysine amino groups become modified and thus converted to amidine derivatives which contain sulfhydryl groups, the modified ribosomal subunits are oxidized to promote crosslinking by formation of intermolecular disulfide bonds. The proteins are extracted and subjected to polyacrylamide/dodecyl sulfate disc gel electrophoresis under non-reducing conditions. The gel is immersed in a reducing solution and then embedded in a second polyacrylamide/dodecyl sulfate gel slab for electrophoresis in a second dimension. Non-cross- linked proteins retain their relative mobility in the two electrophoretic steps and fall on a diagonal line. Proteins crosslinked by disulfide bonds migrate slowly in the first electrophoretic separation but, following reduction, give rise to faster migrating monomeric proteins which appear beneath the diagonal in the second electrophoretic separation. Crosslinked proteins can be identified by their position on the gel pattern and by analysis of the apparent molecular weights of crosslinked species and monomeric proteins derived from them upon reduction. The use of the reversible protein crosslinking procedure and two-dimensional “diagonal” gel electrophoresis provides a characteristic fingerprint of the protein:protein interactions in ribosomal subunits. These techniques, developed to study the ribosome, should be valuable in investigations of other biological ultrastructures in which it is useful to obtain information concerning the arrangement of multiple protein components.


Disulfide Bond Ribosomal Protein Sulfhydryl Group Ribosomal Subunit Protein Pair 


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Copyright information

© Plenum Press, New York 1977

Authors and Affiliations

  • Robert R. Traut
    • 1
  • James W. Kenny
    • 1
  1. 1.Department of Biological Chemistry, School of MedicineUniversity of CaliforniaDavisUSA

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