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. 1993 Oct;2(10):1732–1748. doi: 10.1002/pro.5560021017

Disulfide structures of highly bridged peptides: a new strategy for analysis.

W R Gray 1
PMCID: PMC2142270  PMID: 8251945

Abstract

A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide chain be split between close or adjacent Cys residues. The water-soluble tris-(2-carboxyethyl)-phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol-disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol-disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra- and interchain disulfides; -Cys.Cys- pair), endothelin and apamin (two disulfides; -Cys.x.Cys- pair), conotoxin GI and isomers (two disulfides; -Cys.Cys- pair), and bacterial enterotoxin (three bridges within 13 residues; two -Cys.Cys- pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.

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Selected References

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  1. Brown J. R., Hartley B. S. Location of disulphide bridges by diagonal paper electrophoresis. The disulphide bridges of bovine chymotrypsinogen A. Biochem J. 1966 Oct;101(1):214–228. doi: 10.1042/bj1010214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Callewaert G. L., Shipolini R., Vernon C. A. The disulphide bridges of apamin. FEBS Lett. 1968 Aug;1(2):111–113. doi: 10.1016/0014-5793(68)80033-x. [DOI] [PubMed] [Google Scholar]
  3. Creighton T. E. Interactions between cysteine residues as probes of protein conformation: the disulphide bond between Cys-14 and Cys-38 of the pancreatic trypsin inhibitor. J Mol Biol. 1975 Aug 25;96(4):767–776. doi: 10.1016/0022-2836(75)90151-5. [DOI] [PubMed] [Google Scholar]
  4. Fischer W. H., Rivier J. E., Craig A. G. In situ reduction suitable for matrix-assisted laser desorption/ionization and liquid secondary ionization using tris(2-carboxyethyl)phosphine. Rapid Commun Mass Spectrom. 1993 Mar;7(3):225–228. doi: 10.1002/rcm.1290070312. [DOI] [PubMed] [Google Scholar]
  5. Goldenberg D. P. Native and non-native intermediates in the BPTI folding pathway. Trends Biochem Sci. 1992 Jul;17(7):257–261. doi: 10.1016/0968-0004(92)90405-x. [DOI] [PubMed] [Google Scholar]
  6. Gray W. R. Echistatin disulfide bridges: selective reduction and linkage assignment. Protein Sci. 1993 Oct;2(10):1749–1755. doi: 10.1002/pro.5560021018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gray W. R., Luque F. A., Galyean R., Atherton E., Sheppard R. C., Stone B. L., Reyes A., Alford J., McIntosh M., Olivera B. M. Conotoxin GI: disulfide bridges, synthesis, and preparation of iodinated derivatives. Biochemistry. 1984 Jun 5;23(12):2796–2802. doi: 10.1021/bi00307a040. [DOI] [PubMed] [Google Scholar]
  8. Gray W. R., Rivier J. E., Galyean R., Cruz L. J., Olivera B. M. Conotoxin MI. Disulfide bonding and conformational states. J Biol Chem. 1983 Oct 25;258(20):12247–12251. [PubMed] [Google Scholar]
  9. Hidaka Y., Sato K., Nakamura H., Kobayashi J., Ohizumi Y., Shimonishi Y. Disulfide pairings in geographutoxin I, a peptide neurotoxin from Conus geographus. FEBS Lett. 1990 May 7;264(1):29–32. doi: 10.1016/0014-5793(90)80756-9. [DOI] [PubMed] [Google Scholar]
  10. Hoeprich P. D., Jr, Doolittle R. F. Dimeric half-molecules of human fibrinogen are joined through disulfide bonds in an antiparallel orientation. Biochemistry. 1983 Apr 26;22(9):2049–2055. doi: 10.1021/bi00278a003. [DOI] [PubMed] [Google Scholar]
  11. Houghten R. A., Ostresh J. M., Klipstein F. A. Chemical synthesis of an octadecapeptide with the biological and immunological properties of human heat-stable Escherichia coli enterotoxin. Eur J Biochem. 1984 Nov 15;145(1):157–162. doi: 10.1111/j.1432-1033.1984.tb08535.x. [DOI] [PubMed] [Google Scholar]
  12. Kumagaye S., Kuroda H., Nakajima K., Watanabe T. X., Kimura T., Masaki T., Sakakibara S. Synthesis and disulfide structure determination of porcine endothelin: an endothelium-derived vasoconstricting peptide. Int J Pept Protein Res. 1988 Dec;32(6):519–526. doi: 10.1111/j.1399-3011.1988.tb01383.x. [DOI] [PubMed] [Google Scholar]
  13. Levison M. E., Josephson A. S., Kirschenbaum D. M. Reduction of biological substances by water-soluble phosphines: gamma-globulin (IgG). Experientia. 1969 Feb 15;25(2):126–127. doi: 10.1007/BF01899076. [DOI] [PubMed] [Google Scholar]
  14. Nishiuchi Y., Kumagaye K., Noda Y., Watanabe T. X., Sakakibara S. Synthesis and secondary-structure determination of omega-conotoxin GVIA: a 27-peptide with three intramolecular disulfide bonds. Biopolymers. 1986;25 (Suppl):S61–S68. [PubMed] [Google Scholar]
  15. Nishiuchi Y., Sakakibara S. Primary and secondary structure of conotoxin GI, a neurotoxic tridecapeptide from a marine snail. FEBS Lett. 1982 Nov 8;148(2):260–262. doi: 10.1016/0014-5793(82)80820-x. [DOI] [PubMed] [Google Scholar]
  16. Pink J. R., Milstein C. Inter heavy-light chain disulphide bridge in immune globulins. Nature. 1967 Apr 1;214(5083):92–94. doi: 10.1038/214092a0. [DOI] [PubMed] [Google Scholar]
  17. RYLE A. P., SANGER F. Disulphide interchange reactions. Biochem J. 1955 Aug;60(4):535–540. [PMC free article] [PubMed] [Google Scholar]
  18. RYLE A. P., SANGER F., SMITH L. F., KITAI R. The disulphide bonds of insulin. Biochem J. 1955 Aug;60(4):541–556. doi: 10.1042/bj0600541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Raschdorf F., Dahinden R., Maerki W., Richter W. J., Merryweather J. P. Location of disulphide bonds in human insulin-like growth factors (IGFs) synthesized by recombinant DNA technology. Biomed Environ Mass Spectrom. 1988 Oct;16(1-12):3–8. doi: 10.1002/bms.1200160102. [DOI] [PubMed] [Google Scholar]
  20. Rivier J., Galyean R., Gray W. R., Azimi-Zonooz A., McIntosh J. M., Cruz L. J., Olivera B. M. Neuronal calcium channel inhibitors. Synthesis of omega-conotoxin GVIA and effects on 45Ca uptake by synaptosomes. J Biol Chem. 1987 Jan 25;262(3):1194–1198. [PubMed] [Google Scholar]
  21. Rivier J., McClintock R., Galyean R., Anderson H. Reversed-phase high-performance liquid chromatography: preparative purification of synthetic peptides. J Chromatogr. 1984 Apr 24;288(2):303–328. doi: 10.1016/s0021-9673(01)93709-4. [DOI] [PubMed] [Google Scholar]
  22. Rüegg U. T., Rudinger J. Reductive cleavage of cystine disulfides with tributylphosphine. Methods Enzymol. 1977;47:111–116. doi: 10.1016/0076-6879(77)47012-5. [DOI] [PubMed] [Google Scholar]
  23. SPACKMAN D. H., STEIN W. H., MOORE S. The disulfide bonds of ribonuclease. J Biol Chem. 1960 Mar;235:648–659. [PubMed] [Google Scholar]
  24. Shimonishi Y., Hidaka Y., Koizumi M., Hane M., Aimoto S., Takeda T., Miwatani T., Takeda Y. Mode of disulfide bond formation of a heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli. FEBS Lett. 1987 May 4;215(1):165–170. doi: 10.1016/0014-5793(87)80134-5. [DOI] [PubMed] [Google Scholar]
  25. Yanagisawa M., Kurihara H., Kimura S., Tomobe Y., Kobayashi M., Mitsui Y., Yazaki Y., Goto K., Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988 Mar 31;332(6163):411–415. doi: 10.1038/332411a0. [DOI] [PubMed] [Google Scholar]
  26. Zhang R. M., Snyder G. H. Dependence of formation of small disulfide loops in two-cysteine peptides on the number and types of intervening amino acids. J Biol Chem. 1989 Nov 5;264(31):18472–18479. [PubMed] [Google Scholar]
  27. Zhang R. M., Snyder G. H. Factors governing selective formation of specific disulfides in synthetic variants of alpha-conotoxin. Biochemistry. 1991 Nov 26;30(47):11343–11348. doi: 10.1021/bi00111a021. [DOI] [PubMed] [Google Scholar]
  28. Zhou Z. R., Smith D. L. Location of disulfide bonds in antithrombin III. Biomed Environ Mass Spectrom. 1990 Dec 5;19(12):782–786. doi: 10.1002/bms.1200191206. [DOI] [PubMed] [Google Scholar]

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