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Isopeptide Linkage Between N-A-Monomethylalanine and Lysine

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Isopeptide Linkage Between N-A-Monomethylalanine and Lysine Proc. Nati. Acad. Sci. USA Vol. 74, No. 11, pp. 4905-4908, November 1977 Biochemistry Isopeptide linkage between N-a-monomethylalanine and lysine in ribosomal protein S 1 from Escherichia coli (ribosome structure/protein modification/dansyl amino acids/solid-state peptide synthesis) ROBERT CHEN* AND URSULA CHEN-SCHMEISSER Max-Planck-Institut fur Molekulare Genetik, Abt. Wittmann, D-1000 Berlin-Dahlem, Germany Communicated by Stanford Moore, August 29, 1977 ABSTRACT Protein SI1 from the Escherichia coli ribosome peptides were digested with the enzyme (100:1, by weight) in has a unique NHrterminal structure not previously observed 0.2 M N-methylmorpholine, pH 8.1, for 1 hr at 37'. Amino acid among ribosomal proteins. Owing to the formation of an iso- analyses were performed under standard conditions (12) on a peptide bond between a secondary amino acid (N-a-mono- methylalanine) and the eamino group of the NH2-terminal ly- Durrum D500 amino acid analyzer. sine residue, a "branching point" is formed. Therefore, two Sequence Determinations. The NH2-terminal sequence of amino acids are seen when the NH2 terminus of the protein is the intact protein was determined by the automatic Edman determined. degradation process (13) (model PS 110, Socosi, Paris, France). The thiazolinone derivatives of the amino acids were converted Protein biosynthesis is a result of a number of highly coordi- to the phenylthiohydantoins in 1 M HCI at 800 for 10 min and nated events in which the proper codon-anticodon interaction extracted from the aqueous solution with ethyl acetate. They between mRNA and aminoacylated tRNAs guarantees the were identified by chromatography on silica gel thin-layer correct translation of the genetic message (reviewed in refs. 1 plates (HPTLC silica gel6o, 10 X 10, Merck, Darmstadt, GFR) and 2). This interaction is governed by several ribosomal in system I [chloroform/1-propanol/2-propanol, 98:1:1 (vol/ components, one of which is protein S1i from the Escherichia vol)] and in system II [dichloroethane/acetic acid, 6:1 (vol/vol)]. coli ribosome. This protein has been shown to have a strong In addition, they were hydrolyzed in HI to generate the free influence on the fidelity of translation (3). amino acids (14) and were then subjected to amino acid analysis. Recently the occurrence of an unusual modification, N-a- The refined micro dansyl-Edman technique (10) was used to monomethylation, of the NH2-terminal alanine in Sil was establish the sequences of the tryptic peptides. Micro polyamide reported (4). This residue was determined by mass spectrom- plates were photographed with Agfa-ortho film. etry, amino acid analysis, and chromatography in various sys- Synthesis of the Tripeptide L-Lys(eN-methyl-L-Ala)LAla. tems. In a search for similarly modified ribosomal proteins, The tripeptide was synthesized in solid phase attached to a N-a-monomethylation was also established for the NH2 termini chloromethyl resin (15). After the synthesis was completed the of proteins L16 (4, 5) and L33 (4, 6, 7). The NH2-terminal peptide was cleaved from the resin with HBr in trifluoroacetic residues of these proteins are completely methylated (4). N- acid. It was purified with the micro Dowex 50W-X7 column a-Monomethylation was not measured for protein Si 1 because system (11). Its homogeneity was judged by analytical peptide the Edman degradation process always indicated the presence maps (10), NH2 terminus determination (16) and amino acid of lysine in addition to the presence of N-a-monomethylalanine analysis (12). t-Butoxycarbonyl (Boc)-alanine and a-L-benzy- (NaMeAla). This suggested some "heterogeneity" in the pro- loxycarbonyl (Cbz)-eBoc-L-lysine were purchased from Serva tein. The experiments reported in this communication were (Heidelberg, GFR). Boc-NaMeAla was synthesized with t- designed to investigate this observation. butylazidoformate and was recrystallized from ethyl acetate/ hexane (17). MATERIALS AND METHODS Protein SlI used throughout these experiments was provided RESULTS by H. G. Wittmann. Its identity and purity were judged NHrTerminal Sequence of SlI. Determination of the NH2 by one-dimensional sodium dodecyl sulfate/gel electrophoresis terminus of protein SIl by the dansylation technique gave one (8) and two-dimensional polyacrylamide gel electrophoresis spot in the position of dansyl (Dns)-NaMeAla and a second spot (9). in the position of a- or eDns-lysine on the polyamide plate. This Preparation of Peptides. Protein Si1 was digested with second spot is always seen when proteins (or peptides) that trypsin (TPCK-trypsin, Merck, Darmstadt, GFR). The peptides contain lysine residues react with dansyl chloride. This is due were purified by peptide mapping on cellulose thin-layer plates to the reactivity of the e-amino group of lysine. Bis-Dns-lysine or by ion exchange chromatography on a Dowex 50W-X7 is seen when lysine occurs at the NH2 terminus of the protein micro column (10, 11). A more detailed report of the prepara- (or peptide). Therefore, it was concluded that the sample tion of the peptides will appear elsewhere. contained pure protein SIl and that the NH2-terminal residue Amino Acid Analyses. Hydrolyses of the peptides were was NaMeAla. performed in 6 M HCI in the presence of 0.02% 2-mqrcapto- This conclusion, however, conflicted with the data from the ethanol or by digestion with leucine aminopeptidase (Serva, Heidelberg, GFR). Amounts corresponding to 200 pmol of the Abbreviations: NaMeAla, N-a-monomethylalanine; Boc, t-butoxy- carbonyl; Cbz, benzyloxycarbonyl; Dns, dansyl (1-dimethylamino- The costs of publication of this article were defrayed in part by the naphthalene-5-sulfonyl); PhNCS, 3-phenyl-2-thiohydantoin; PhNHCS, payment of page charges. This article must therefore be hereby marked phenylthiocarbamyl. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate * Present address: Max-Planck-Institut ffir Biologie, Corrensstrasse 38, this fact. D-7400 Tubingen 1, Germany. 4905 Downloaded by guest on September 24, 2021 4906 Biochemistry: Chen and Chen-Schmeisser Proc. Natl. Acad. Sci. USA 74 (1977) Table 1. Amino acid composition of the NH2-terminal tryptic peptide of S11 1 NMA p Amino Standard amino Calibration with acid acid analysis Lys(E-N-MeAla)-Ala # S > 3* I NaMeAla + 1.10 A * A Ala 1.10 0.95 o K 6***t * t Ile 1.00 &.. Pro 0.95 Lys 1.01 1.00 I * * 9~~~~~~~R R R Arg 0.92 O A.- V.**ae..+A. STEP: 1 2 3 4 5 6 7 8 addition, it revealed that the second amino group of lysine FIG.....1. Automatic * Edman degradation of S11. The phenylthio- became accessible to dansyl chloride when the sample was hydantoin (PhNCS)*A*..-....derivatives of the amino acids released in every hydrolyzed before it was treated with the reagent. step of the analysis were identified on silica gel thin-layer plates. Standard PhNCS-amino acid mixtures were chromatographed par- Analysis with Leucine Aminopeptidase. Digestion with allel to them (18). Note that two amino acids are seen in every step. leucine aminopeptidase was used to determine whether one A = Ala; I = Ile; K = Lys; P = Pro; R = Arg; NMA = NaMeAla. amino group of the NH2-terminal lysine in Sli was blocked by alkylation, acylation, or glycosylation, because leucine automatic degradation of Sil in the sequenator (Fig. 1). In the aminopeptidase would not hydrolyze these residues from lysine. first step of the analysis, both NaMeAla and lysine were re- The NH2-terminal tryptic peptide, which contained a single leased from the protein. Two amino acids also were seen in each lysine residue, was treated with the enzyme. From the digest of the following steps, indicating that apparently two poly- an aliquot was dansylated and chromatographed on polyamide peptide chains were present in the sample. In addition, the data plates, which revealed that both bis-Dns-lysine and Dns- suggested, that the "two" chains were homologous, because an NaMeAla were present on the chromatogram. This result amino acid seen in one step of the analysis also occurred in the showed that alkylation, acylation, or glycosylation of the lysine next step. Hence, the difference between the "two" chains residue had to be excluded. seemed to be an additional amino acid at the NH2 terminus of Measurement of NaMeAla in the NH2-Terminal Tryptic one of them that induced a shift of the entire sequence. Peptide. No standard amino acid mixtures are available that Sequence of the NH2-Terminal Tryptic Peptide. To resolve contain NaMeAla. Therefore, the synthetic peptide L-Lys(E- the discrepancy between the dansylation results and the se- N-methyl-L-Ala)-L-Ala was used to calibrate the absorptivity quenator data, protein SlI was digested with trypsin. The of NaMeAla relative to equimolar amounts of lysine and ala- NH2-terminal tryptic peptide was isolated from the digest on nine. Amino acid analyses of both the synthetic peptide and the peptide maps. This peptide was sequenced by the micro dan- NH2-terminal tryptic peptide of Si 1 were conducted on a syl-Edman technique which gave Dns-NaMeAla and a- or Durrum D500 amino acid analyzer at the sensitivity of 2 OD, e-Dns-lysine in the first cycle (Fig. 2). Bis-Dns-lysine was not at which the standard error of the analysis is minimal. The data seen in the first step nor was lysine identified in any further step are given in Table 1. The molar ratio of NaMeAla to lysine and of the analysis. alanine was the same in both peptides, which showed that In order to investigate why a- or E-Dns-lysine was seen rather NaMeAla and lysine occurred in every molecule of the than bis-Dns-lysine in the first cycle, the NH2-terminal tryptic NH2-terminal tryptic peptide. The data confirmed that the peptide was submitted to a subtractive micro dansyl-Edman peptide was pure and that we were not dealing with a mixture technique.
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