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. 2001 Dec;81(6):3522–3533. doi: 10.1016/S0006-3495(01)75983-8

Structural characterization of the pH-denatured states of ferricytochrome-c by synchrotron small angle X-ray scattering.

S Cinelli 1, F Spinozzi 1, R Itri 1, S Finet 1, F Carsughi 1, G Onori 1, P Mariani 1
PMCID: PMC1301807  PMID: 11721013

Abstract

The ferricytochrome-c (cyt-c) shows a complex unfolding pathway characterized by a series of stable partially folded states. When titrated with HCl at low ionic strength, two transitions are detected. At pH 2, cyt-c assumes the U1 unfolded state, whereas the successive addition of Cl(-) ion from either HCl or NaCl induces the recompaction to a molten globule conformation (A1 and A2 states, respectively). A second unfolded state (U2) is also observed at pH 12. Recent data evidence different features for the local structure of the heme in the different states. To derive relationships between local and overall conformations, we analyzed the structural characteristics of the different states by synchrotron small angle X-ray scattering. The results show that in the acidic-unfolded U1 form the protein assumes a worm-like conformation, whereas in the alkaline-unfolded U2 state, the cyt-c is globular. Moreover, the molten globule states induced by adding HCl or NaCl to U1 appear structurally different: in the A1 state cyt-c is dimeric and less compact, whereas in the A2 form the protein reverts to a globular-like conformation. According to the local heme structure, a molecular model for the different forms is derived.

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Selected References

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  1. Ashton A. W., Boehm M. K., Gallimore J. R., Pepys M. B., Perkins S. J. Pentameric and decameric structures in solution of serum amyloid P component by X-ray and neutron scattering and molecular modelling analyses. J Mol Biol. 1997 Sep 26;272(3):408–422. doi: 10.1006/jmbi.1997.1271. [DOI] [PubMed] [Google Scholar]
  2. Banci L., Bertini I., Huber J. G., Spyroulias G. A., Turano P. Solution structure of reduced horse heart cytochrome c. J Biol Inorg Chem. 1999 Feb;4(1):21–31. doi: 10.1007/s007750050285. [DOI] [PubMed] [Google Scholar]
  3. Boffi F., Bonincontro A., Cinelli S., Congiu Castellano A., De Francesco A., Della Longa S., Girasole M., Onori G. pH-dependent local structure of ferricytochrome c studied by x-ray absorption spectroscopy. Biophys J. 2001 Mar;80(3):1473–1479. doi: 10.1016/S0006-3495(01)76119-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Christensen H., Pain R. H. Molten globule intermediates and protein folding. Eur Biophys J. 1991;19(5):221–229. doi: 10.1007/BF00183530. [DOI] [PubMed] [Google Scholar]
  5. Fink A. L., Calciano L. J., Goto Y., Kurotsu T., Palleros D. R. Classification of acid denaturation of proteins: intermediates and unfolded states. Biochemistry. 1994 Oct 18;33(41):12504–12511. doi: 10.1021/bi00207a018. [DOI] [PubMed] [Google Scholar]
  6. Fink A. L. Compact intermediate states in protein folding. Annu Rev Biophys Biomol Struct. 1995;24:495–522. doi: 10.1146/annurev.bb.24.060195.002431. [DOI] [PubMed] [Google Scholar]
  7. Goto Y., Calciano L. J., Fink A. L. Acid-induced folding of proteins. Proc Natl Acad Sci U S A. 1990 Jan;87(2):573–577. doi: 10.1073/pnas.87.2.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goto Y., Takahashi N., Fink A. L. Mechanism of acid-induced folding of proteins. Biochemistry. 1990 Apr 10;29(14):3480–3488. doi: 10.1021/bi00466a009. [DOI] [PubMed] [Google Scholar]
  9. Henderson S. J. Monte Carlo modeling of small-angle scattering data from non-interacting homogeneous and heterogeneous particles in solution. Biophys J. 1996 Apr;70(4):1618–1627. doi: 10.1016/S0006-3495(96)79725-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jeng M. F., Englander S. W., Elöve G. A., Wand A. J., Roder H. Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR. Biochemistry. 1990 Nov 20;29(46):10433–10437. doi: 10.1021/bi00498a001. [DOI] [PubMed] [Google Scholar]
  11. Kataoka M., Hagihara Y., Mihara K., Goto Y. Molten globule of cytochrome c studied by small angle X-ray scattering. J Mol Biol. 1993 Feb 5;229(3):591–596. doi: 10.1006/jmbi.1993.1064. [DOI] [PubMed] [Google Scholar]
  12. Kataoka M., Nishii I., Fujisawa T., Ueki T., Tokunaga F., Goto Y. Structural characterization of the molten globule and native states of apomyoglobin by solution X-ray scattering. J Mol Biol. 1995 May 26;249(1):215–228. doi: 10.1006/jmbi.1995.0290. [DOI] [PubMed] [Google Scholar]
  13. Mariani P., Carsughi F., Spinozzi F., Romanzetti S., Meier G., Casadio R., Bergamini C. M. Ligand-induced conformational changes in tissue transglutaminase: Monte Carlo analysis of small-angle scattering data. Biophys J. 2000 Jun;78(6):3240–3251. doi: 10.1016/S0006-3495(00)76860-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ohgushi M., Wada A. 'Molten-globule state': a compact form of globular proteins with mobile side-chains. FEBS Lett. 1983 Nov 28;164(1):21–24. doi: 10.1016/0014-5793(83)80010-6. [DOI] [PubMed] [Google Scholar]
  15. Pollack L., Tate M. W., Darnton N. C., Knight J. B., Gruner S. M., Eaton W. A., Austin R. H. Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle x-ray scattering. Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):10115–10117. doi: 10.1073/pnas.96.18.10115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pérez J., Vachette P., Russo D., Desmadril M., Durand D. Heat-induced unfolding of neocarzinostatin, a small all-beta protein investigated by small-angle X-ray scattering. J Mol Biol. 2001 May 11;308(4):721–743. doi: 10.1006/jmbi.2001.4611. [DOI] [PubMed] [Google Scholar]
  17. Semisotnov G. V., Kihara H., Kotova N. V., Kimura K., Amemiya Y., Wakabayashi K., Serdyuk I. N., Timchenko A. A., Chiba K., Nikaido K. Protein globularization during folding. A study by synchrotron small-angle X-ray scattering. J Mol Biol. 1996 Oct 4;262(4):559–574. doi: 10.1006/jmbi.1996.0535. [DOI] [PubMed] [Google Scholar]
  18. Stuhrmann H. B., Koch M. H., Parfait R., Haas J., Ibel K., Crichton R. R. Shape of the 50S subunit of Escherichia coli ribosomes. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2316–2320. doi: 10.1073/pnas.74.6.2316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Svergun D. I., Richard S., Koch M. H., Sayers Z., Kuprin S., Zaccai G. Protein hydration in solution: experimental observation by x-ray and neutron scattering. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2267–2272. doi: 10.1073/pnas.95.5.2267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tanford C. Protein denaturation. C. Theoretical models for the mechanism of denaturation. Adv Protein Chem. 1970;24:1–95. [PubMed] [Google Scholar]
  21. Trewhella J. Insights into biomolecular function from small-angle scattering. Curr Opin Struct Biol. 1997 Oct;7(5):702–708. doi: 10.1016/s0959-440x(97)80081-4. [DOI] [PubMed] [Google Scholar]
  22. Weber C., Michel B., Bosshard H. R. Spectroscopic analysis of the cytochrome c oxidase-cytochrome c complex: circular dichroism and magnetic circular dichroism measurements reveal change of cytochrome c heme geometry imposed by complex formation. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6687–6691. doi: 10.1073/pnas.84.19.6687. [DOI] [PMC free article] [PubMed] [Google Scholar]

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