The Sulfur Chemistry of Proteins

Cecil, R.; McPhee, J.R.

doi:10.1016/s0065-3233(08)60613-0

Cited by 323 publications

(151 citation statements)

References 709 publications

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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%

“…The larger fragment was identified by N-terminal sequencing and MS to be comprised of residues 1-58, and it contained the first six half-Cystines (C7, C20, C32, C26, C38, and C55). This fragment was isolated in two forms, a single-Chain polypeptide comprised of residues 1-58 (found m/z ¼ 6133.5, Table I), and its hetero-dimeric product, residues [1][2][3][4][5][6][7][8] to , resulting from cleavage of the peptide bond Trp8-Asn9 (Table I). The smaller fragment, identified by N-terminal sequencing and MS to be comprised of residues 59-95, contained the remaining four half-Cystines paired in two disulfides (C68, C80, C74, C86; found m/z ¼ 3592.4).…”

Section: Limited Proteolysis Of Svn With Pepsinmentioning

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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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Limited Proteolysis Of Svn With Pepsinmentioning

confidence: 99%

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 second ionizing group (pK approx. 9.5) may be attributed to the enzyme, (Cecil & McPhee, 1959). Support for this hypothesis comes from the fact that the enzyme is inhibited by a number of reagents known to react with thiol groups (Mattock & Jones, 1970).…”

Section: Methodsmentioning

confidence: 99%

The effect of substrate concentration and pH on the enzymic sulphation of l-tyrosyl derivatives

Mattock¹,

Barford²,

Basford³

et al. 1970

Biochemical Journal

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1. The kinetics of the enzymic transfer of sulphate from adenosine 3'-phosphate 5'[35S]-sulphatophosphate to derivatives of L-tyrosine were investigated with a partially purified enzyme preparation from rat liver. 2. At pH 7.5 and 370C the Km values for L-tyrosine methyl ester and adenosine 3'-phosphate 5'[35S]-sulphatophosphate are 0.3mM and 8nM respectively. The Km value for either substrate is independent of the concentration of the other. The available data are consistent with the sulphation reaction proceeding according to a rapid-equilibrium random Bi Bi mechanism. 3. From the effect of pH on the Km and Vmax. values for L-tyrosine methyl ester, tyramine and N-acetyl-L-tyrosine ethyl ester it is concluded that the enzyme is specific for substrate molecules with a free and unprotonated amino group and an un-ionized hydroxyl group. 4. The only ionizing group that can be positively attributed to the enzyme appears to influence the binding of adenosine 3'-phosphate 5'[35S]-sulphatophosphate and has an apparent pK value of approx. 9.5. It is suggested that this group may be an essential thiol. 5. The enzyme is inhibited by iodoacetamide at pH 7.5 and 30°C and this inhibition is prevented by the presence of adenosine 3'-phosphate 5'[35S]-sulphatophosphate but not by L-tyrosine methyl ester.The preceding paper (Mattock & Jones, 1970) contrasted some of the properties of the enzymes responsible for the sulphation of L-tyrosine methyl ester and tyramine on the one hand and p-nitrophenol on the other. From the evidence quoted, it appears that the two enzymes are distinct from one another. Hence it is not likely that the appearance of L-tyrosine 0-sulphate in mammalian urines (John, Rose, Wusteman & Dodgson, 1966) is an artifact of a detoxication type of reaction. However, whether the enzyme that catalyses the sulphation of simple derivatives of L-tyrosine is responsible for the appearance of sulphated tyrosine residues in proteins is unknown, and an investigation of the substrate requirements of this enzyme may throw light on its possible metabolic function. In this connexion, Segal & Mologne (1959) have shown that, with a rat liver enzyme preparation, the rate of transfer of sulphate from p-nitrophenyl sulphate via adenosine 3',5'-diphosphate to carboxyl-substituted derivatives of L-tyrosine was higher at pH 9.3 than at pH 7.8. These authors suggested that these observations reflected the ionization of the amino group of the substrates in

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Site‐directed Spin Labeling and Pulse Dipolar Electron Paramagnetic Resonance

Klare

Steinhoff

2010

Encyclopedia of Analytical Chemistry

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Structural and functional investigations on biomolecules often rely on the availability of topological information, either to allow building structural models or to characterize conformational changes of these molecules or complexes thereof during function. Site‐directed spin labeling (SDSL) in combination with EPR spectroscopy became a very powerful tool to obtain intra‐ and inter‐molecular distance constraints, as the accessible distances span the interesting range from 0.5–8 nm, crystallization is not required, and the size of the system under investigation is not limited. Especially the development of pulse dipolar EPR methods, in particular double electron–electron resonance (DEER) spectroscopy with its ability to provide interspin distance distributions in the 2–8 nm range, greatly increased the applicability of electron paramagnetic resonance (EPR) spectroscopy for studies on biological systems. This article is intended to give an introduction into SDSL and dipolar EPR. The first part summarizes the state‐of‐the‐art in SDSL, providing an overview of methodologies currently available or under development. In the second part, the technique of DEER spectroscopy is introduced, covering the basic theoretical background as well as selected practical aspects of data acquisition and analysis. Finally, selected examples for the application of SDSL‐DEER from the recent literature are summarized.

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The Sulfur Chemistry of Proteins

Cited by 323 publications

References 709 publications

Topology of the disulfide bonds in the antiviral lectin scytovirin

Topology of the disulfide bonds in the antiviral lectin scytovirin

The effect of substrate concentration and pH on the enzymic sulphation of l-tyrosyl derivatives

Site‐directed Spin Labeling and Pulse Dipolar Electron Paramagnetic Resonance

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