Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Oct 15;335(Pt 2):343–349. doi: 10.1042/bj3350343

Susceptibility towards intramolecular disulphide-bond formation affects conformational stability and folding of human basic fibroblast growth factor.

D Estapé 1, J van den Heuvel 1, U Rinas 1
PMCID: PMC1219788  PMID: 9761733

Abstract

The conformational stability and the folding properties of the all-beta-type protein human basic fibroblast growth factor (hFGF-2) were studied by means of fluorescence spectroscopy. The results show that the instability of the biological activity of hFGF-2 is also reflected in a low conformational stability of the molecule. The reversibility of the unfolding and refolding process was established under reducing conditions. Determination of the free-energy of unfolding in the presence of reducing agents revealed that the conformational stability of hFGF-2 (DeltaGH2Oapp congruent with21 kJ. mol-1, 25 degreesC) is low compared with other globular proteins under physiological conditions (20-60 kJ.mol-1). However, the conformational stability of hFGF-2 is particularly low under non-reducing conditions. This instability is attributed to intramolecular disulphide-bond formation, rendering the molecule more susceptible to denaturant-induced unfolding. In addition, denaturant-induced unfolding of hFGF-2 renders the protein more susceptible to irreversible oxidative denaturation. Experimental evidence is provided that the irreversibility of the unfolding and refolding process in the absence of reducing agents is linked to the formation of an intramolecular disulphide bond involving cysteines 96 and 101.

Full Text

The Full Text of this article is available as a PDF (634.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ago H., Kitagawa Y., Fujishima A., Matsuura Y., Katsube Y. Crystal structure of basic fibroblast growth factor at 1.6 A resolution. J Biochem. 1991 Sep;110(3):360–363. doi: 10.1093/oxfordjournals.jbchem.a123586. [DOI] [PubMed] [Google Scholar]
  2. Ahmad F., Bigelow C. C. Estimation of the free energy of stabilization of ribonuclease A, lysozyme, alpha-lactalbumin, and myoglobin. J Biol Chem. 1982 Nov 10;257(21):12935–12938. [PubMed] [Google Scholar]
  3. Arakawa T., Hsu Y. R., Schiffer S. G., Tsai L. B., Curless C., Fox G. M. Characterization of a cysteine-free analog of recombinant human basic fibroblast growth factor. Biochem Biophys Res Commun. 1989 May 30;161(1):335–341. doi: 10.1016/0006-291x(89)91601-x. [DOI] [PubMed] [Google Scholar]
  4. Buntrock P., Buntrock M., Marx I., Kranz D., Jentzsch K. D., Heder G. Stimulation of wound healing, using brain extract with fibroblast growth factor (FGF) activity. III. Electron microscopy, autoradiography, and ultrastructural autoradiography of granulation tissue. Exp Pathol. 1984;26(4):247–254. doi: 10.1016/s0232-1513(84)80057-2. [DOI] [PubMed] [Google Scholar]
  5. Buntrock P., Jentzsch K. D., Heder G. Stimulation of wound healing, using brain extract with fibroblast growth factor (FGF) activity. II. Histological and morphometric examination of cells and capillaries. Exp Pathol. 1982;21(1):62–67. doi: 10.1016/s0232-1513(82)80054-6. [DOI] [PubMed] [Google Scholar]
  6. Burgess W. H., Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem. 1989;58:575–606. doi: 10.1146/annurev.bi.58.070189.003043. [DOI] [PubMed] [Google Scholar]
  7. Caccia P., Nitti G., Cletini O., Pucci P., Ruoppolo M., Bertolero F., Valsasina B., Roletto F., Cristiani C., Cauet G. Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues. Eur J Biochem. 1992 Mar 1;204(2):649–655. doi: 10.1111/j.1432-1033.1992.tb16678.x. [DOI] [PubMed] [Google Scholar]
  8. Eriksson A. E., Cousens L. S., Matthews B. W. Refinement of the structure of human basic fibroblast growth factor at 1.6 A resolution and analysis of presumed heparin binding sites by selenate substitution. Protein Sci. 1993 Aug;2(8):1274–1284. doi: 10.1002/pro.5560020810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eriksson A. E., Cousens L. S., Weaver L. H., Matthews B. W. Three-dimensional structure of human basic fibroblast growth factor. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3441–3445. doi: 10.1073/pnas.88.8.3441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fox G. M., Schiffer S. G., Rohde M. F., Tsai L. B., Banks A. R., Arakawa T. Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine. J Biol Chem. 1988 Dec 5;263(34):18452–18458. [PubMed] [Google Scholar]
  11. Gospodarowicz D., Cheng J. Heparin protects basic and acidic FGF from inactivation. J Cell Physiol. 1986 Sep;128(3):475–484. doi: 10.1002/jcp.1041280317. [DOI] [PubMed] [Google Scholar]
  12. Gospodarowicz D., Ferrara N., Schweigerer L., Neufeld G. Structural characterization and biological functions of fibroblast growth factor. Endocr Rev. 1987 May;8(2):95–114. doi: 10.1210/edrv-8-2-95. [DOI] [PubMed] [Google Scholar]
  13. Greene R. F., Jr, Pace C. N. Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, alpha-chymotrypsin, and beta-lactoglobulin. J Biol Chem. 1974 Sep 10;249(17):5388–5393. [PubMed] [Google Scholar]
  14. Iwane M., Kurokawa T., Sasada R., Seno M., Nakagawa S., Igarashi K. Expression of cDNA encoding human basic fibroblast growth factor in E. coli. Biochem Biophys Res Commun. 1987 Jul 31;146(2):470–477. doi: 10.1016/0006-291x(87)90553-5. [DOI] [PubMed] [Google Scholar]
  15. Kallis G. B., Holmgren A. Differential reactivity of the functional sulfhydryl groups of cysteine-32 and cysteine-35 present in the reduced form of thioredoxin from Escherichia coli. J Biol Chem. 1980 Nov 10;255(21):10261–10265. [PubMed] [Google Scholar]
  16. Meyer-Ingold W. Wound therapy: growth factors as agents to promote healing. Trends Biotechnol. 1993 Sep;11(9):387–392. doi: 10.1016/0167-7799(93)90098-T. [DOI] [PubMed] [Google Scholar]
  17. Nelson J. W., Creighton T. E. Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo. Biochemistry. 1994 May 17;33(19):5974–5983. doi: 10.1021/bi00185a039. [DOI] [PubMed] [Google Scholar]
  18. Nozaki Y., Tanford C. The solubility of amino acids, diglycine, and triglycine in aqueous guanidine hydrochloride solutions. J Biol Chem. 1970 Apr 10;245(7):1648–1652. [PubMed] [Google Scholar]
  19. Pace C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986;131:266–280. doi: 10.1016/0076-6879(86)31045-0. [DOI] [PubMed] [Google Scholar]
  20. Pace C. N. The stability of globular proteins. CRC Crit Rev Biochem. 1975 May;3(1):1–43. doi: 10.3109/10409237509102551. [DOI] [PubMed] [Google Scholar]
  21. Robinson C. J. Growth factors in wound healing. Trends Biotechnol. 1992 Sep;10(9):301–302. doi: 10.1016/0167-7799(92)90253-r. [DOI] [PubMed] [Google Scholar]
  22. Santoro M. M., Bolen D. W. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry. 1988 Oct 18;27(21):8063–8068. doi: 10.1021/bi00421a014. [DOI] [PubMed] [Google Scholar]
  23. Seeger A., Rinas U. Two-step chromatographic procedure for purification of basic fibroblast growth factor from recombinant Escherichia coli and characterization of the equilibrium parameters of adsorption. J Chromatogr A. 1996 Oct 4;746(1):17–24. doi: 10.1016/0021-9673(96)00286-5. [DOI] [PubMed] [Google Scholar]
  24. Seno M., Sasada R., Iwane M., Sudo K., Kurokawa T., Ito K., Igarashi K. Stabilizing basic fibroblast growth factor using protein engineering. Biochem Biophys Res Commun. 1988 Mar 15;151(2):701–708. doi: 10.1016/s0006-291x(88)80337-1. [DOI] [PubMed] [Google Scholar]
  25. Shing Y., Folkman J., Sullivan R., Butterfield C., Murray J., Klagsbrun M. Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor. Science. 1984 Mar 23;223(4642):1296–1299. doi: 10.1126/science.6199844. [DOI] [PubMed] [Google Scholar]
  26. Sluzky V., Shahrokh Z., Stratton P., Eberlein G., Wang Y. J. Chromatographic methods for quantitative analysis of native, denatured, and aggregated basic fibroblast growth factor in solution formulations. Pharm Res. 1994 Apr;11(4):485–490. doi: 10.1023/a:1018946011652. [DOI] [PubMed] [Google Scholar]
  27. Snyder G. H., Cennerazzo M. J., Karalis A. J., Field D. Electrostatic influence of local cysteine environments on disulfide exchange kinetics. Biochemistry. 1981 Nov 10;20(23):6509–6519. doi: 10.1021/bi00526a001. [DOI] [PubMed] [Google Scholar]
  28. Tardieu M., Bourin M. C., Desgranges P., Barbier P., Barritault D., Caruelle J. P. Mesoglycan and sulodexide act as stabilizers and protectors of fibroblast growth factors (FGFs). Growth Factors. 1994;11(4):291–300. doi: 10.3109/08977199409011002. [DOI] [PubMed] [Google Scholar]
  29. Thompson S. A., Fiddes J. C. Chemical characterization of the cysteines of basic fibroblast growth factor. Ann N Y Acad Sci. 1991;638:78–88. doi: 10.1111/j.1749-6632.1991.tb49019.x. [DOI] [PubMed] [Google Scholar]
  30. Thompson S. A., Protter A. A., Bitting L., Fiddes J. C., Abraham J. A. Cloning, recombinant expression, and characterization of basic fibroblast growth factor. Methods Enzymol. 1991;198:96–116. doi: 10.1016/0076-6879(91)98012-u. [DOI] [PubMed] [Google Scholar]
  31. Thompson S. A. The disulfide structure of bovine pituitary basic fibroblast growth factor. J Biol Chem. 1992 Feb 5;267(4):2269–2273. [PubMed] [Google Scholar]
  32. Vemuri S., Beylin I., Sluzky V., Stratton P., Eberlein G., Wang Y. J. The stability of bFGF against thermal denaturation. J Pharm Pharmacol. 1994 Jun;46(6):481–486. doi: 10.1111/j.2042-7158.1994.tb03831.x. [DOI] [PubMed] [Google Scholar]
  33. Weich H. A., Iberg N., Klagsbrun M., Folkman J. Expression of acidic and basic fibroblast growth factors in human and bovine vascular smooth muscle cells. Growth Factors. 1990;2(4):313–320. doi: 10.3109/08977199009167026. [DOI] [PubMed] [Google Scholar]
  34. Westall F. C., Rubin R., Gospodarowicz D. Brain-derived fibroblast growth factor: a study of its inactivation. Life Sci. 1983 Dec 12;33(24):2425–2429. doi: 10.1016/0024-3205(83)90636-7. [DOI] [PubMed] [Google Scholar]
  35. Zhang J. D., Cousens L. S., Barr P. J., Sprang S. R. Three-dimensional structure of human basic fibroblast growth factor, a structural homolog of interleukin 1 beta. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3446–3450. doi: 10.1073/pnas.88.8.3446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhu X., Komiya H., Chirino A., Faham S., Fox G. M., Arakawa T., Hsu B. T., Rees D. C. Three-dimensional structures of acidic and basic fibroblast growth factors. Science. 1991 Jan 4;251(4989):90–93. doi: 10.1126/science.1702556. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES