β‐Methylthio‐aspartic acid: Identification of a novel posttranslational modification in ribosomal protein S12 from escherichia coli

Abstract

Utilizing microscale chemical derivatization reactions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we have identified a novel posttranslational modification of aspartic acid, j3-methylthio-aspartic acid. The modified residue is located at position 88 in ribosomal protein S12 from Escherichia coli, a phylogenetically conserved protein that has been implicated in maintaining translational accuracy of the ribosome.Keywords: j3-methylthio-aspartic acid; MALDI-TOF; postsource decay; posttranslational modification As a research tool, mass spectrometry has become an integral component in modern biochemistry, where it has been useful in elucidation of the primary structures of all classes of biopolymers, and especially in the characterization of posttranscriptional and posttranslational modifications. Mass spectrometry techniques are inherently advantageous because the intrinsic property of molecular mass provides objective chemical information as an element of the characterization of modified residues (for example, see Biemann, 1988Biemann, , 1990, and references therein). Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (Hillenkamp et al., 1991) offers the additional advantage of adhering to criteria of exceptionally low detection limits (femtomoles to picomoles), low sample consumption (typically I pL) and compatibility with a wide range of buffer components, while generating high quality molecular weight information. Furthermore, valuable sequence informa- During the course of examining Escherichia coli ribosomal proteins isolated from 30s subunits by MALDI-TOF MS, it was observed that the molecular mass of protein S12 was 46 Da heavier that that predicted from the gene sequence (Post & Nomura, 1980). Inspection of the literature revealed that the amino acid at residue 88 in S12 had not been identified (Funatsu Utilizing microscale chemical derivatization techniques (Knapp, 1979(Knapp, , 1990) and relying heavily on MALDI-TOF MS for molecular weight information and PSD for peptide sequencing, we now describe the identification of a novel posttranslationally modified amino acid, 0-methylthio-aspartic acid, at position 88 in ribosomal protein S12 from E. coli. ResultsE. coli ribosomal proteins were isolated from purified 30s subunits and fractionated by reversed-phase HPLC. The molecular mass of ribosomal protein Si2 was measured to be 13,652.0Da by MALDI-TOF MS (Fig. 1). Using electrospray ionization mass spectrometry, two species were observed: a minor one (approximately lo%), M , , 13,605.2; and a major one, M2r 13,652.1 Da (data not shown). From the corresponding gene sequence (Post & Nomura, 1980), the expected mass was of S12 calculated to be 13,605.9 Da. These data suggest posttranscriptional modification of S12 as indicated by a mass increment of 46 Da. It is not clear whether the unmodified form of S12 is a 1625

Post-translational Modification of Ribosomal Proteins

“…8, A and B). This methylthiolation is likely to occur on the 3-carbon position of the aspartate residue as suggested previously (4). The MS2 of compound 2 failed to localize the second methylthiolation.…”

“…These modifications are believed to extend molecular structures beyond the limits imposed by the 20 genetically encoded amino acids (2). For example, the Escherichia coli ribosomal protein S12 is shown to be post-translationally modified through 3-methylthiolation of the Asp-89 3 residue (Scheme 1A), a modification believed to improve translational accuracy (3,4). Recently, the yliG gene (later named rimO for ribosomal modification O) has been shown to be responsible for this reaction in vivo (5).…”

Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins

“…The requirement for ordered addition of the recombinant proteins for efficient reconstitution could be due to a number of possible factors+ Some of the small subunit ribosomal proteins are subject to posttranslational modification (Leibowitz & Soffer, 1971;Cumberlidge & Isono, 1979;Reeh & Pedersen, 1979;Isono & Isono, 1980;Kowalak & Walsh, 1996)+ Therefore, one or more of the overproduced, recombinant proteins might be substoichiometrically modified+ Although all of the recombinant proteins were overexpressed in E. coli and thus available to their natural modification enzymes, the levels of expression may exceed the capacity of the endogenous enzymes+ Ordered assembly could obviate the need for specific modifications; binding a subset of proteins to 16S rRNA could promote or increase the lifetime of transiently formed intermediates, thus allowing an inadequately modified protein, perhaps with a weakened binding affinity, the opportunity to interact productively with its target, prior to the addition of other proteins and subsequent conformational changes+ Proteins that were isolated from ribosomes may be in a more functional conformation if prior ribosome assembly involves rearrangement of protein structure; therefore, it is possible that one or more of the recombinant proteins is incompletely folded as isolated, and that other proteins and/or RNA could stimulate their folding into a more functional conformation+ Thus, ordered assembly could help this folding problem if incubating the incompletely folded protein with a subset of small subunit proteins or 16S rRNA potentiates folding+ This could reflect the process that occurs in vivo, where a subset of proteins might initiate assembly cotranscriptionally+ Either of the above possibilities could result in overestimation of the concentration of functional protein+ The same would be true if a subpopulation of protein(s) were inactivated during purification, as was hypothesized for proteins isolated from ribosomes (Nomura et al+, 1969)+ A complete set of recombinant small subunit ribosomal proteins was produced with the hope of generating an easily renewable source of large quantities of highly purified individual proteins, obviating the difficult and laborious purification of individual proteins from ribosomal subunits+ This set of proteins will be of great use, not only in studying the proteins themselves but, also as tools for studying the structure, function, and assembly of 30S subunits and 70S ribosomes+ Along with the ability to purify tens of milligrams of ribosomal proteins with relative ease, the high levels of overexpression of these proteins can provide proteins that are essentially free of contamination with other ribosomal and cellular proteins+ Finally, systematic mutational analysis of the small subunit ribosomal proteins is facilitated by the availability of useful clones encoding all of their genes+…”

Analysis of the Arabidopsis Cytosolic Ribosome Proteome Provides Detailed Insights into Its Components and Their Post-translational Modification

“…In addition to heterogeneity derived from different ribosomal genes for the same subunit, covalent modification introduces further potential differences in ribosomal protein structure and function. Covalent modifications of ribosomal proteins have been reported in bacteria (28,29,(31)(32)(33)(34)(35), fungi (3, 4, 8, 36 -38), mammals (5)(6)(7)39), and plants (19). However, in almost all of these studies, limitations of the techniques used, such as peptide mass fingerprinting, topdown MS analysis of intact r-proteins, and acid hydrolysis to remove rRNA, have precluded detection of acid/base-labile, low stoichiometry, or difficult-to-detect modifications or the determination of precise details about the exact position(s) and structure(s) of the modified residue(s).…”