The formation of dehydroalanine residues in alkali-treated insulin and oxidized glutathione. A nuclear-magnetic-resonance study

Jones, Anthony; Helmerhorst, Erik; Stokes, Gilbert B.

doi:10.1042/bj2110499

Cited by 26 publications

(20 citation statements)

References 10 publications

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“…These possibilities can be distinguished by NMR spectroscopy. Both Dha and Dhb contain vinyl protons, which typically give resonance peaks in the ␦ ϭ 5.2-6.9 ppm region of the NMR spectrum, with Dha appearing as a doublet, and Dhb appearing as a quartet (7,(21)(22)(23). The NMR spectrum of sublancin is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification and Characterization of the Structural and Transporter Genes for, and the Chemical and Biological Properties of, Sublancin 168, a Novel Lantibiotic Produced by Bacillus subtilis 168

Paik

Chakicherla

Hansen

1998

Journal of Biological Chemistry

225

241

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An antimicrobial peptide produced by Bacillus subtilis 168 was isolated and characterized. It was named sublancin 168, and its behavior during Edman sequence analysis and its NMR spectrum suggested that sublancin is a dehydroalanine-containing lantibiotic. A hybridization probe based on the peptide sequence was used to clone the presublancin gene, which encoded a 56-residue polypeptide consisting of a 19-residue leader segment and a 37-residue mature segment. The mature segment contained one serine, one threonine, and five cysteine residues. Alkylation of mature sublancin showed no free sulfhydryl groups, suggesting that one sulfydryl had formed a ␤-methyllanthionine bridge with a dehydrobutyrine derived by posttranslational modification of threonine; with the other four cysteines forming two disulfide bridges. It is unprecedented for a lantibiotic to contain a disulfide bridge. The sublancin leader was similar to known type AII lantibiotics, containing a double-glycine motif that is typically recognized by dual-function transporters. A protein encoded immediately downstream from the sublancin gene possessed features of a dual-function ABC transporter with a proteolytic domain and an ATP-binding domain. The antimicrobial activity spectrum of sublancin was like other lantibiotics, inhibiting Gram-positive bacteria but not Gram-negative bacteria; and like the lantibiotics nisin and subtilin in its ability to inhibit both bacterial spore outgrowth and vegetative growth. Sublancin is an extraordinarily stable lantibiotic, showing no degradation or inactivation after being stored in aqueous solution at room temperature for 2 years. The fact that sublancin is a natural product of B. subtilis 168, for which a great deal of genetic information is available, including the entire sequence of its genome, suggests that sublancin will be an especially good model for studying the potential of lantibiotics as sources of novel biomaterials.

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Section: Resultsmentioning

confidence: 99%

Identification and Characterization of the Structural and Transporter Genes for, and the Chemical and Biological Properties of, Sublancin 168, a Novel Lantibiotic Produced by Bacillus subtilis 168

Paik

Chakicherla

Hansen

1998

Journal of Biological Chemistry

225

241

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“…The proposed fragmentation pathways for the formation of dehydroalanine, thioaldehyde, and reduced glutathione are reminiscent of chemical cleavages occurring at the cystine site in the peptide under base catalyzed condition in solutions [23][24][25]. We have attempted to obtain experimental support for these proton abstraction processes by subjecting a deuterated sample of glutathione, in which all exchangeable hydrogens have been replaced by deuterium, to collision induced dissociation in an ion trap mass spectrometer.…”

Section: Contryphanmentioning

confidence: 99%

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

Thakur

Balaram

2008

J. Am. Soc. Mass Spectrom.

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The fragmentation of positive and negative ions of peptide disulfides under mass spectrometric conditions yields distinctly different product ion distributions. A negative ion upon collision induced dissociation yields intense product ions, which correspond to cleavage at the disulfide linkage. The complete assignment of the product ions obtained upon fragmentation of oxidized glutathione in an ion trap is presented. The cleavage at the disulfide site is mediated by abstraction of C ␣ H and C ␤ p H protons resulting in product ions derived by neutral loss of H 2 S 2 and H 2 S. The formation of peptide thioaldehydes and persulfides at the cysteine sites is established. Dehydroalanine formation at the Cys residue is predominant. The case of a contryphan, a cyclic peptide disulfide derived from Conus snail venom, illustrates the utility of negative ion mass spectrometry in disulfide identification. M ass spectrometric characterization of disulfide bonded peptides is generally achieved by the reduction of the S-S bond followed by alkylation [1][2][3][4] or by oxidation to sulfonic acid derivatives [5]. Subsequent gas-phase fragmentation results in readily identifiable backbone cleavage products. Several groups have focused on the mass spectrometric analysis of peptides containing intact disulfide bonds using both positive and negative ions for gas-phase fragmentation [6 -12]. In the case of positively charged peptide ions, disulfide bond fragmentation occurs with much lower efficiency than cleavage of backbone peptide bonds, under conditions of collision induced dissociation with non-mobile protons suggested to play an important role [11]. Restricted proton mobility along the polypeptide backbone has also been implicated in cases of disulfide cleavages in polypeptides, which have been cationized using gold (I) [7]. The use of metal ions as gas-phase disulfide cleavage agents has also been investigated [12]. In general, only limited information regarding structure can be derived from intact disulfide peptides in the positive ion mode. In contrast, several recent studies have emphasized the utility of negative ion mass spectrometry [7,10,[13][14][15][16][17][18]. Specifically, Bowie and coworkers have presented assignments of product ion spectra obtained by negative ion electrospray mass spectrometry of peptides containing both intra and inter molecular disulfide bond [10,18]. The present study complements the work of Bowie and coworkers and further provides mechanistic details by examining second generation product ions by using ion trap mass spectrometry.In the structural analysis of peptides containing intact disulfides, under negative ion conditions, proton abstraction from the C ␣ H and C ␤ H positions of the Cys residue results in diverse fragmentation reactions, leading to cleavage of disulfide linkages. In the case of peptides that contain labile peptide bonds, for example X-Pro sequences, preferential cleavage at these sites results in two linear chains linked by a disulfide bond, which then undergo further f...

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“…The topology of disulfides in SVN was originally assigned by matching the masses of its tryptic fragments obtained by LC-MS, 1 as well as of fragments of expressed SD1. 2,5 Starting with assignment of species ionizing as m/z ¼ 1318.6 (residues 30-43; C32-C38; SD1), m/z ¼ 1553.6 (residues 79-95; C80-C86; SD2), and m/z ¼ 3157.5 (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] to [51-59]; C7-55), the remaining two species, termed here ''cystine clusters'' of m/z ¼ 2511.0 and m/z ¼ 2719.1, each containing two disulfides, C20-C26/C32-C38 (SD1) and C68-C74/C80-C86 (SD2), were assigned by process of elimination.…”

Section: Resultsmentioning

confidence: 99%

“…8 and 9 ). At alkaline pH, cystine beta-elimination, and desulfurization with subsequent formation of alternative cross-links (such as the lanthionine thioether links) are common 8,10,11 and observable by modern mass spectrometry techniques. 12,13 However, unlike cystine beta-elimination/desulfurization, cystine interchange is more difficult to trace by current analytical approaches and has long been recognized as the confounding factor in protein structural analyses; if not controlled or minimized, artifactual assignments may ensue.…”

Section: Discussionmentioning

confidence: 99%

“…As can be seen from fragment assignments [ Fig. 2(A), Table II], a heterodimeric peptide, residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]-[51-60], containing C7-C55, has been identified (fragment 10). Its Edman degradation yielded two N-terminal sequences, including di-PTH-Cystine in Cycle 7, confirming the presence of a disulfide bridge.…”

Section: Assignment Of the C7-c55 Disulfide Bondmentioning

confidence: 99%

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Topology of the disulfide bonds in the antiviral lectin scytovirin

et al. 2010

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The antiviral lectin scytovirin (SVN) contains a total of five disulfide bonds in two structurally similar domains. Previous reports provided contradictory results on the disulfide pairing in each individual domain, and we have now re-examined the disulfide topology. N-terminal sequencing and mass spectrometry were used to analyze proteolytic fragments of native SVN obtained at acidic pH, yielding the assignment as Cys7-Cys55, Cys20-Cys32, Cys26-Cys38, Cys68-Cys80, and Cys74-Cys86. We also analyzed the N-terminal domain of SVN (SD1, residues 1-48) prepared by expression/oxidative folding of the recombinant protein and by chemical synthesis. The disulfide pairing in the chemically synthesized SD1 was forced into predetermined topologies: SD1A (Cys20-Cys26, Cys32-Cys38) or SD1B (Cys20-Cys32, Cys26-Cys38). The topology of native SVN was found to be in agreement with the SD1B and the one determined for the recombinant SD1 domain. Although the two synthetic forms of SD1 were distinct when subjected to chromatography, their antiviral properties were indistinguishable, having low nM activity against HIV. Tryptic fragments, the ''cystine clusters'' [Cys20-Cys32/Cys26-Cys38; SD1] and [Cys68-Cys80/ Disclaimer: The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.Abbreviations: Acm, acetamidomethyl; ATZ, 2-anilino-5-thiazolinone; AUFS, absorbance unit full scale; Boc-Gly-OCH 2 -PAM, t-butyloxycarbonyl-glycyl-4-(hydroxymethyl)phenylacetamidomethyl-copoly(styrene/1% divinylbenzene) resin; DIEA, diisopropylethylamine; ESI-Q-TOF, electrospray ionization quadrupole-time-of-flight mass spectrometry; HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino) methylene]-N-methyl-methanaminium hexafluorophosphate N-oxide; HFBA, heptafluorobutyric acid; MALDI-TOF, matrix-assisted laser desorption-ionization time-of-flight; MS/MS, tandem mass spectrometry; m/z, mass-to-charge ratio; pE, pyroglutamyl; PTH, phenylthiohydantoin; RP-HPLC, reversed-phase high pressure liquid chromatography; rSD1, recombinant/oxidatively folded N-terminal domain of SVN (residues 1-48Cys7Ser); R.T., retention time on HPLC; SD1A, synthetic N-terminal domain of SVN [residues 1-48Cys7Ser (Cys20-Cys26, Cys32-Cys38)]; SD1B, synthetic N-terminal domain of SVN [residues 1-48Cys7Ser (Cys20-Cys32, Cys26-Cys38)]; TCEP, tris(2-Carboxyethyl)phosphine; TFA, trifluoroacetic acid. Cys74-C-86; SD2], were found to undergo rapid disulfide interchange at pH 8. This interchange resulted in accumulation of artifactual fragments in alkaline pH digests that are structurally unrelated to the original topology, providing a rational explanation for the differences between the topology reported herein and the one reported earlier (Bokesh et al., Biochemistry 2003;42:2578-2584. Our observations emphasize the fact that proteins such as SVN, with disulfide bonds in close proximity, require consider...

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The formation of dehydroalanine residues in alkali-treated insulin and oxidized glutathione. A nuclear-magnetic-resonance study

Cited by 26 publications

References 10 publications

Identification and Characterization of the Structural and Transporter Genes for, and the Chemical and Biological Properties of, Sublancin 168, a Novel Lantibiotic Produced by Bacillus subtilis 168

Identification and Characterization of the Structural and Transporter Genes for, and the Chemical and Biological Properties of, Sublancin 168, a Novel Lantibiotic Produced by Bacillus subtilis 168

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

Topology of the disulfide bonds in the antiviral lectin scytovirin

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