Determination of the Complete Amino-Acid Sequence of Protein S6 from the Wild-Type and a Mutant of Escherichia coli

Abstract

Protein S6 could be isolated in five forms of different length and acidity from the mixture of all 70-S ribosomal proteins by column chromatography on DEAE-cellulose followed by purification on Sephadex G-100. Amino acid sequence studies on the intact protein and a fragment obtained using 2-(2-nitrophenyl-sulfenyl)-3-methyl-3′-bromoindolenine skatole were done by automatic Edman degradation using an improved liquid-phase Beckman sequenator. The protein was digested with trypsin and all 19 peptides were isolated. Their amino acid compositions were analysed and the sequence of the amino acids within the peptides was determined either by means of a solid-phase peptide sequenator or by using the dansyl-Edman technique. Further, protein S6 was digested with chymotrypsin, thermolysin, staphylococcal protease, trypsin after modification of lysyl ɛ-amino groups by exo-cis-3,6-endoxo-Δ4-tetrahydrophthalic acid anhydride and with 2-(2-nitrophenyl-sulfenyl)-3-methyl-3′-bromoindolenine skatole, a reagent which specifically cleaves tryptophanyl bonds. Amino acid analyses and partial sequencing of these fragments established the alignment of the tryptic peptides. The longest form of S6 (S6-6) consists of 135 amino acids and has an isoelectric point of pH 4.8. The shorter forms S6-5, S6-4, S6-3 and S6-2 show no difference in their amino acid composition and sequence with the exception that they have one, two, three and four glutamic acid residues less, respectively, at their C-termini. A comparison of the amino acid sequence of S6 with that of the most acidic protein L12 from the 50-S subunit and other ribosomal proteins of known primary structure is made. Further a prediction for regions with α-helices and with β-sheets in protein S6 is performed. The amino acid sequence of a mutationally altered S6 protein was shown to be identical with the primary structure of S6-2, the shortest form of wild-type protein S6.