Enhancement of Cyanogen Bromide Cleavage Yields for Methionyl-Serine and Methionyl-Threonine Peptide Bonds

Kaiser, R. E.; Metzka, Lorraine

doi:10.1006/abio.1998.2945

Cited by 105 publications

(72 citation statements)

References 14 publications

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“…While it is unclear if this hydration was caused in the synthesis of the reporter or in the subsequent peptide mapping assay, since the absorbance at 338 nm was sufficient to detect the key peptides, it was not investigated further. Additionally, the unexhaustive peptide harboring the PCP1 active site serine displayed a -46 Da species, which has been observed in prior mechanistic studies of cyanogen bromide digestion (21). This modification is likely due to a side reaction during digestion, where Thr 1336 is involved in the formation of a second fivemembered ring, ultimately permitting ejection of methyl thiocyanate without cleaving the amide bond.…”

Section: Discussionmentioning

confidence: 59%

“…In this work, we describe the implementation of a cyanogen bromide digestion protocol and the use of several coenzyme A analogues with spectroscopic, ∆ mass, and affinity properties for mapping the modified active site peptides from the pyochelin synthetase biosynthetic enzymes, PchE (159 kDa) and PchF (198 kDa) ( Figures 6 and 7 of the Supporting Information) (20)(21)(22). These "reporter"-coenzyme A analogues were exclusively loaded onto the conserved active site serines within the thiolation domains of PchE and PchF using the indiscriminate 4′-phosphopantetheinyl transferase, Sfp (23).…”

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confidence: 99%

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Chemoenzymatic Approaches for Streamlined Detection of Active Site Modifications on Thiotemplate Assembly Lines Using Mass Spectrometry

et al. 2005

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For the direct interrogation of peptides harboring covalently modified serines in nonribosomal peptide synthetases, streamlined methodologies described here employ proteolysis and reporter-coenzyme A analogues of four types. The chromophoric and fluorescent coenzyme A analogues pyrene-maleimidyl-S-CoA and BODIPY-FL-N-(2-aminoethyl)maleimidyl-S-CoA were enzymatically loaded onto the active site serines harbored in the ArCP, PCP1, and PCP2 thiolation domains of PchE and PchF, the nonribosomal peptide synthetases responsible for the biosynthesis of the siderophore pyochelin. During the chromatographic separation of cyanogen bromide digests, observation of the absorbance (at 338 and 504 nm) or fluorescence (after irradiation at 365 nm) enabled the selective detection of peptides containing each active site serine. This resulted in quick detection of each active site peptide by Fourier transform mass spectrometry in the fully reconstituted pyochelin system. The loading of short acyl chain reporters in equimolar quantities permitted further insights into digestion heterogeneity and side reactions by virtue of a mass shift signature on each active site peptide. The chromatographic shift of the reporter-loaded peptides relative to peptides carrying on pathway intermediates was 2 min at 7 kDa, providing a general strategy for efficient localization of "carrier" peptides in complex digests of thiotemplate enzymes. Also, the use of the affinity reporter, biotin-maleimidyl-S-coenzyme A, permitted the isolation of intact synthetases at high purity via removal of contaminating Escherichia coli proteins.

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Section: Discussionmentioning

confidence: 59%

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confidence: 99%

Chemoenzymatic Approaches for Streamlined Detection of Active Site Modifications on Thiotemplate Assembly Lines Using Mass Spectrometry

et al. 2005

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“…Purified fusion protein was concentrated using an Amicon Ultra-15 centrifugal filter devices (Millipore) and exchanged into 50 mM sodium phosphate buffer (pH 7) containing 300 mM NaCl and 1 mM PMSF and diluted in TFA to a final concentration of 70% TFA. Fifteen microliters of 5 M CNBr solution, which is a common chemical used to hydrolyze peptide bonds C-terminal to methionine residues in peptides and proteins [12], was added to 1.1 ml of the 70% TFA solution which was protected from light and allowed to incubate for 17 h at room temperature. After CNBr cleavage the mixture was diluted in 20 ml of 50 mM sodium phosphate buffer (pH 7) containing 300 mM NaCl and 1 mM PMSF followed by concentration and buffer-exchange against 50 mM sodium phosphate buffer (pH 6.8) containing 1 mM PMSF.…”

Section: Protein Expression Purification and Sample Preparationmentioning

confidence: 99%

Self‐recognition by an intrinsically disordered protein

et al. 2008

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The intrinsically disordered translocation domain (T-domain) of the protein antibiotic colicin N binds to periplasmic receptors of target Escherichia coli cells in order to penetrate their inner membranes. We report here that the specific 27 consecutive residues of the T-domain of colicin N known to bind to the helper protein TolA in target cells also interacts intramolecularly with folded regions of colicin N. We suggest that this specific self-recognition helps intrinsically disordered domains to bury their hydrophobic recognition motifs and protect them against degradation, showing that an impaired self-recognition leads to increased protease susceptibility.

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“…The mutant protein was overexpressed in E. coli and treated with CNBr to cleave the presequence after the introduced methionine. During the cleavage reaction the methionine was converted into a homoserine lactone (hsl) group to generate N 5.7 pF 1 ␤-(2-54)-hsl (50). Purification of N 5.7 pF 1 ␤-(2-54)-hsl was performed as described above for N 15 pF 1 ␤.…”

Section: Purification Of a Novel Metalloprotease From Solanum Tuberosummentioning

confidence: 99%

Isolation and Identification of a Novel Mitochondrial Metalloprotease (PreP) That Degrades Targeting Presequences in Plants

Ståhl¹,

Moberg²,

Ytterberg³

et al. 2002

Journal of Biological Chemistry

116

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Most of the nuclear encoded mitochondrial precursor proteins contain an N-terminal extension called the presequence that carries targeting information and that is cleaved off after import into mitochondria. The presequences are amphiphilic, positively charged, membrane-interacting peptides with a propensity to form ␣-helices. Here we have investigated the proteolysis of the presequences that have been cleaved off inside mitochondria. A presequence derived from the overexpressed F 1 ␤ subunit of the ATP synthase and specific synthetic fluorescent peptides (Pep Tag Protease assay) have been shown to undergo rapid degradation catalyzed by a matrix located protease. We have developed a three-step chromatographic procedure including affinity and anion exchange chromatography for isolation of the protease from potato tuber mitochondria. Twodimensional gel electrophoresis of the isolated proteolytically active fraction followed by electrospray ionization-mass spectrometry/mass spectrometry and data base searches allowed identification of the presequence peptide-degrading protease in Arabidopsis thaliana data base as a novel mitochondrial metalloendoprotease with a molecular mass of 105 kDa. The identified metalloprotease contains an inverted zinc-binding motif and belongs to the pitrilysin family.

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Enhancement of Cyanogen Bromide Cleavage Yields for Methionyl-Serine and Methionyl-Threonine Peptide Bonds

Cited by 105 publications

References 14 publications

Chemoenzymatic Approaches for Streamlined Detection of Active Site Modifications on Thiotemplate Assembly Lines Using Mass Spectrometry

Chemoenzymatic Approaches for Streamlined Detection of Active Site Modifications on Thiotemplate Assembly Lines Using Mass Spectrometry

Self‐recognition by an intrinsically disordered protein

Isolation and Identification of a Novel Mitochondrial Metalloprotease (PreP) That Degrades Targeting Presequences in Plants

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