Identification of a cross-link in the Escherichia coli ribosomal protein pair S13-S19 at the amino acid level.
Escherichia coli 30 S ribosomal subunits and 70 S ribosomes were treated with the bifunctional reagent diepoxybutane, acting as a cross-linker. One major cross-linked protein pair in the 30 S subunit was generated in relatively high yields. This cross-link was shown to consist of ribosomal proteins S13 and S19. Purification of this complex was achieved by a series of conventional and/or high pressure liquid chromatography techniques allowing its isolation in milligram quantities. To reveal the exact position of the two amino acids involved in the cross-link formation, the purified protein pair S13-S19 was subjected to several enzymatic fragmentations, and the resulting peptides were characterized by sequence analysis, amino acid analysis, and fast atom bombardment mass spectrometry. After isolation of the cross-linked peptides, Cys84 in protein S13 and His68 in S19 could be unequivocally identified as the amino acids cross-linked by the bifunctional reagent. This result demonstrates that, despite neutron scattering data which place the centers of mass of S13 and S19 85 A apart, at least these regions of the two proteins are located within a 4-A distance in the ribosomal particle.
Pohl
T
T
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin Dahlem, West Germany.
Wittmann-Liebold
B
B
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D012269:Ribosomal Proteins / Q000032:analysis*
D000596:Amino Acids; D029968:Escherichia coli Proteins; D012269:Ribosomal Proteins; C040343:ribosomal protein S19; C512838:rpsM protein, E coli