Identification of the domains of disulfide interaction in the mammalian ribosomal proteins L6 and L29

Sammanfattning: The 60-S subunits of mammalian ribosomes contain two adjacent proteins, identified as L6 and L29, which interact reversibly in situ by disulfide bond formation. The disulfide complex and the individual proteins were prepared by polyacrylamide gel electrophoresis under non-oxidizing conditions for effective prevention of methionine oxidation. Characterization of the proteins by HPLC-peptide mapping revealed only few sequence homologies. Further analysis indicated that the interacting thiols represent the only cysteine groups present in the two proteins. By cyanylation and alkaline cleavage the distance of these interacting groups from the C-and N- terminal ends of the two chains was determined. This provided a favorable opportunity for the first time to identify and characterize the contact domains of two contiguous ribosomal proteins.The interacting domains were isolated and characterized by cyanogen bromide, chymotryptic, peptic and S. aureus protease cleavage of the isolated complex and the '4c-carboxymethylated proteins. The linear arrangement of the CNBr fragments was established by microscale, in-gel methodology using the inherently recognizable thiol groups a specific reference points for the alignment.After determining the location of the interacting thiols on the polypeptide chains, the structural organization of the proteins on the ribosome was studied using trypsin, clostripain and S. aureus protease as surface probe. The combined experiments indicate that the site of interaction of L6 occurs approx 80 amino acid residues from the N-terminus. In intact ribosomes this part contains a terminal sequence of 30-38 amino acid residues exposed at the ribosomal surface, as indicated by its great susceptibility to hydrolysis in situ by the two enzymes. On the basis of these data it is concludecTthat the site of interaction of protein L6 is located about 42-50 amino acid residues inside the exposed part of the molecule. Protein L29 has a more external location in the particle structure. Its reactive thiol is located approx 40 amino acid residues from the C-terminus.The cysteine-containing region of L29 is most likely also structurally shielded as indicated by the restricted accessibility of the thiol group to sulfhydryl reagents.