A Top-Down/Bottom-Up Study of the Ribosomal Proteins of <i>Caulobacter crescentus</i>

Running, William E.; Ravipaty, Shobha; Karty, Jonathan A.; Reilly, James P.

doi:10.1021/pr060306q

Cited by 31 publications

(60 citation statements)

References 83 publications

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“…Proteomic studies of bacterial ribosomal proteins have reported di-or monomethylation at this residue in E. coli (29), Caulobacter crescentus (28), and Rhodopseudomonas palustris (27), and the reported molecular mass of Thermus thermophilus RpL7/L12 also suggests methylation (26). However, this modification appears not to be present in all bacteria as Bacillus subtilis RpL7/L12 was found to be unmodified (45).…”

Section: Discussionmentioning

confidence: 99%

The METTL20 Homologue from Agrobacterium tumefaciens Is a Dual Specificity Protein-lysine Methyltransferase That Targets Ribosomal Protein L7/L12 and the β Subunit of Electron Transfer Flavoprotein (ETFβ)

Małecki

Dahl

Moen

et al. 2016

Journal of Biological Chemistry

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Human METTL20 is a mitochondrial, lysine-specific methyltransferase that methylates the ␤-subunit of electron transfer flavoprotein (ETF␤). Interestingly, putative METTL20 orthologues are found in a subset of ␣-proteobacteria, including Agrobacterium tumefaciens. Using an activity-based approach, we identified in bacterial extracts two substrates of recombinant METTL20 from A. tumefaciens (AtMETTL20), namely ETF␤ and the ribosomal protein RpL7/L12. We show that AtMETTL20, analogous to the human enzyme, methylates ETF␤ on Lys-193 and Lys-196 both in vitro and in vivo. ETF plays a key role in mediating electron transfer from various dehydrogenases, and we found that its electron transferring ability was diminished by AtMETTL20-mediated methylation of ETF␤. Somewhat surprisingly, AtMETTL20 also catalyzed monomethylation of RpL7/L12 on Lys-86, a common modification also found in many bacteria that lack METTL20. Thus, we here identify AtMETTL20 as the first enzyme catalyzing RpL7/ L12 methylation. In summary, here we have identified and characterized a novel bacterial lysine-specific methyltransferase with unprecedented dual substrate specificity within the seven ␤-strand class of lysine-specific methyltransferases, as it targets two apparently unrelated substrates, ETF␤ and RpL7/L12. Moreover, the present work establishes METTL20-mediated methylation of ETF␤ as the first lysine methylation event occurring in both bacteria and humans.

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Section: Discussionmentioning

confidence: 99%

The METTL20 Homologue from Agrobacterium tumefaciens Is a Dual Specificity Protein-lysine Methyltransferase That Targets Ribosomal Protein L7/L12 and the β Subunit of Electron Transfer Flavoprotein (ETFβ)

Małecki

Dahl

Moen

et al. 2016

Journal of Biological Chemistry

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“…It is clear that each species has a unique complement of modifications, and despite the availability of many genomic sequences, MS analysis of isolated ribosomes remains the only way to completely characterize the spectrum of modifications for a new species. In the proteomic study of Caulobacter crescentus, some modifications similar to those observed in E. coli were observed, as well as some novel species-specific modifications [8]. The ability to map modifications in the ribosomal proteome will permit the study of changes in modifications with growth conditions or genotype, which may elucidate the specific role of ribosomal protein modifications in assembly or translation.…”

Section: Applications Of Mass Spectrometrymentioning

confidence: 91%

“…The composition of the ribosomal proteome from Caulobacter crescentus was determined using a combination of MS analysis of intact proteins and of proteolytic fragments [8]. The masses of most of the ribosomal proteins were determined directly, and sequences were confirmed by MS-MS analysis of tryptic peptides.…”

Section: Applications Of Mass Spectrometrymentioning

confidence: 99%

Biophysical studies of bacterial ribosome assembly

Williamson

2008

Current Opinion in Structural Biology

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SUMMARYThe assembly of the bacterial ribosome involves association of over 50 proteins to three large RNA molecules, and it represents a major metabolic activity for rapidly growing bacteria. The availability of atomic structures of the ribosome and the application of biochemical and biophysical methods have led to rapid progress in understanding the mechanistic details of ribosome assembly. The basic steps required to assemble a ribosome are outlined, and the contributions of mass spectrometry, computational methods, and RNA folding studies in understanding these steps are detailed. This complex process takes place with both sequential and parallel processing that is coordinated to ensure efficient and complete of assembly of ribosomes to meet the demands of cell growth.

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“…Mass spectrometry (MS) has become an indispensable tool for protein sequence mapping [1,2]. This is usually done by using a top-down or a bottom-up proteomic approach [1][2][3][4][5][6][7][8][9][10][11][12]. In the top-down method, a protein ion is dissociated in a tandem mass spectrometer (MS/MS) and the fragment ions generated are interpreted to generate a stretch of amino acid sequence information.…”

mentioning

confidence: 99%

“…However, this is a time-consuming process and there is no guarantee that the peptides produced from the multiple digestions will cover the entire sequence of a protein. In some cases, the combination of top-down and bottom-up methods is used to increase sequence coverage [4,7,12]. …”

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confidence: 99%

Reproducible microwave-assisted acid hydrolysis of proteins using a household microwave oven and its combination with LC-ESI MS/MS for mapping protein sequences and modifications

Wang

2010

J. Am. Soc. Mass Spectrom.

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A new set-up for microwave-assisted acid hydrolysis (MAAH) with high efficiency and reproducibility to degrade proteins into peptides for mass spectrometry analysis is described. It is based on the use of an inexpensive domestic microwave oven and can be used for low volume protein solution digestion. This set-up has been combined with liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI QTOF MS) for mapping protein sequences and characterizing phosphoproteins. It is demonstrated that for bovine serum albumin (BSA), with a molecular mass of about 67,000 Da, 1292 peptides (669 unique sequences) can be detected from a 2 g hydrolysate generated by trifluoroacetic acid (TFA) MAAH. These peptides cover the entire protein sequence, allowing the identification of an amino acid substitution in a natural variant of BSA. It is shown that for a simple phosphoprotein containing one phosphoform, ␤-casein, direct analysis of the hydrolysate generates a comprehensive peptide map that can be used to identify all five known phosphorylation sites. For characterizing a complex phosphoprotein consisting of different phosphoforms with varying numbers of phosphate groups and/or phosphorylation sites, such as bovine ␣ S1 -casein, immobilized metal-ion affinity chromatography (IMAC) is used to enrich the phosphopeptides from the hydrolysate, followed by LC-ESI MS analysis. The MS/MS data generated from the initial hydrolysate and the phosphopeptide-enriched fraction, in combination with MS analysis of the intact protein sample, allow us to reveal the presence of three different phosphoforms of bovine ␣ S1 -casein and assign the phosphorylation sites to each phosphoform with high confidence. (J Am Soc Mass Spectrom 2010, 21, 1573-1587) © 2010 American Society for Mass Spectrometry P rotein sequence mapping is commonly used to study post-translational modifications of a protein or amino acid substitutions from point mutations in the genome. Ideally, the entire amino acid sequence of a protein should be mapped to pinpoint where a modified amino acid or a substitution is located. Mass spectrometry (MS) has become an indispensable tool for protein sequence mapping [1,2]. This is usually done by using a top-down or a bottom-up proteomic approach [1][2][3][4][5][6][7][8][9][10][11][12]. In the top-down method, a protein ion is dissociated in a tandem mass spectrometer (MS/MS) and the fragment ions generated are interpreted to generate a stretch of amino acid sequence information. Sequence coverage by this method is dependent on the nature of the protein, ranging from a few residues to a full sequence [12]. In general, full sequence information is difficult to obtain for a protein with molecular mass of Ͼ20,000 Da [12]. The bottom-up approach is a robust method for protein identification based on sequencing by MS/MS one or more peptides generated by chemical or enzymatic degradation of a protein. For a protein digest, such as that produced by trypsin digestion, one or a few peptides are sequenced, r...

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A Top-Down/Bottom-Up Study of the Ribosomal Proteins of Caulobacter crescentus

Cited by 31 publications

References 83 publications

The METTL20 Homologue from Agrobacterium tumefaciens Is a Dual Specificity Protein-lysine Methyltransferase That Targets Ribosomal Protein L7/L12 and the β Subunit of Electron Transfer Flavoprotein (ETFβ)

The METTL20 Homologue from Agrobacterium tumefaciens Is a Dual Specificity Protein-lysine Methyltransferase That Targets Ribosomal Protein L7/L12 and the β Subunit of Electron Transfer Flavoprotein (ETFβ)

Biophysical studies of bacterial ribosome assembly

Reproducible microwave-assisted acid hydrolysis of proteins using a household microwave oven and its combination with LC-ESI MS/MS for mapping protein sequences and modifications

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