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Biochemistry (Moscow)

, Volume 74, Issue 10, pp 1088–1095 | Cite as

Compact acid-induced state of Clitoria ternatea agglutinin retains its biological activity

  • A. Naeem
  • M. Saleemuddin
  • R. H. KhanEmail author
Article
  • 47 Downloads

Abstract

The effects of pH on Clitoria ternatea agglutinin (CTA) were studied by spectroscopy, size-exclusion chromatography, and by measuring carbohydrate specificity. At pH 2.6, CTA lacks well-defined tertiary structure, as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of 50% native-like secondary structure. The mean residue ellipticity at 217 nm plotted against pH showed a transition around pH 4.0 with loss of secondary structure leading to the formation of an acid-unfolded state. This state is relatively less denatured than the state induced by 6 M guanidine hydrochloride. With a further decrease in pH, this unfolded state regains ∼75% secondary structure at pH 1.2, leading to the formation of the A-state with native-like near-UV CD spectral features. Enhanced 8-anilino-1-naphthalene-sulfonate binding was observed in A-state, indicating a “molten-globule” like conformation with exposed hydrophobic residues. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a compact conformation at low pH. Size-exclusion chromatography shows the presence of a compact intermediate with hydrodynamic size corresponding to a monomer. Thermal denaturation of the native state was cooperative single-step transition and of the A-state was non-cooperative two-step transition. A-State regains 72% of the carbohydrate-binding activity.

Key words

acid-induced unfolding Clitoria ternatea agglutinin carbohydrate binding molten-globule state thermal stability 

Abbreviations

ANS

8-anilino-1-naphthalene-sulfonic acid

A-state

acid induced state

CTA

Clitoria ternatea agglutinin

GdnHCl

guanidine hydrochloride

MRE

mean residue ellipticity

SEC

size-exclusion chromatography

UA

acid unfolded state

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References

  1. 1.
    Kuwajima, K. (1989) Proteins, 6, 87–103.CrossRefPubMedGoogle Scholar
  2. 2.
    Ptitsyn, O. B. (1995) Trends Biochem. Sci., 20, 376–379.CrossRefPubMedGoogle Scholar
  3. 3.
    Goto, Y., and Fink, A. L. (1989) Biochemistry, 28, 945–952.CrossRefPubMedGoogle Scholar
  4. 4.
    Dryden, D., and Weir, M. P. (1991) Biochim. Biophys. Acta, 1078, 94–100.PubMedGoogle Scholar
  5. 5.
    Song, J., Bai, P., Luo, L., and Peng, Z. Y. (2001) Protein Sci., 10, 53–62.Google Scholar
  6. 6.
    Colon, W., and Roder, H. (1996) Nat. Struct. Biol., 3, 1019–1025.CrossRefPubMedGoogle Scholar
  7. 7.
    Barrick, D., Hughson, F. M., and Baldwin, R. L. (1994) J. Mol. Biol., 237, 588–601.CrossRefPubMedGoogle Scholar
  8. 8.
    Jaenicke, R. (1991) Biochemistry, 30, 3147–3161.CrossRefPubMedGoogle Scholar
  9. 9.
    Naeem, A., Khan, A., and Khan, R. H. (2005) Biochem. Biophys. Res. Commun., 331, 1284–1294.CrossRefPubMedGoogle Scholar
  10. 10.
    Ballery, N., Desmadril, M., Minard, P., and Yon, J. M. (1993) Biochemistry, 32, 708–714.CrossRefPubMedGoogle Scholar
  11. 11.
    Matousschek, A., Serrano, L., Meiering, E. M., Bycroft, M., and Ferscht, A. R. (1992) J. Mol. Biol., 224, 837–845.CrossRefGoogle Scholar
  12. 12.
    Naeem, A., Khan, K. A., and Khan, R. H. (2004) Arch. Biochem. Biophys., 432, 79–87.PubMedGoogle Scholar
  13. 13.
    Goto, Y., and Nishikiori, S. (1991) J. Mol. Biol., 222, 679–686.CrossRefPubMedGoogle Scholar
  14. 14.
    Goto, Y., Calciano, L. J., and Fink, A. L. (1990) Proc. Natl. Acad. Sci. USA, 87, 573–577.CrossRefPubMedGoogle Scholar
  15. 15.
    Kumar, D. P., Tiwari, A., and Bhatt, R. (2004) J. Biol. Chem., 279, 32093–32099.CrossRefPubMedGoogle Scholar
  16. 16.
    Naeem, A., Haque, S., and Khan, R. H. (2007) Protein J., 26, 403–413.CrossRefPubMedGoogle Scholar
  17. 17.
    Naeem, A., Ahmad, E., and Khan, R. H. (2007) Int. J. Biol. Macromol., 41, 481–486.CrossRefPubMedGoogle Scholar
  18. 18.
    Davis, B. J. (1964) Ann. N. Y. Acad. Sci., 121, 404–407.CrossRefPubMedGoogle Scholar
  19. 19.
    Lowry, D. H., Rosebrough, N. J., Farr, A. L., and Randal, R. J. (1951) J. Biol. Chem., 193, 265–275.PubMedGoogle Scholar
  20. 20.
    Stryer, L. (1965) J. Mol. Biol., 13, 482–495.CrossRefPubMedGoogle Scholar
  21. 21.
    Stryer, L. (1968) Science, 162, 526–540.CrossRefPubMedGoogle Scholar
  22. 22.
    Semisotnov, G. V., Rodionova, N. A., Razgulyaev, O. I., Uversky, V. N., Gripas, A. F., and Gilmanshin, R. I. (1991) Biopolymers, 31, 119–128.CrossRefPubMedGoogle Scholar
  23. 23.
    Matulis, D., and Lovrien, R. (1998) Biophys. J., 74, 422–429.CrossRefPubMedGoogle Scholar
  24. 24.
    Matulis, D., Baumann, C. G., Bloomfield, U. A., and Lovrien, R. E. (1999) Biopolymers, 49, 451–458.CrossRefPubMedGoogle Scholar
  25. 25.
    Muzammil, S., Kumar, Y., and Tayyab, S. (1999) Eur. J. Biochem., 266, 26–32.CrossRefPubMedGoogle Scholar
  26. 26.
    Pawar, S. A., and Deshpande, V. V. (2000) Eur. J. Biochem., 267, 6331–6338.CrossRefPubMedGoogle Scholar
  27. 27.
    Holzman, T. E., Dougherty, J. J., Brems, D. N., and Mackenzie, N. E. (1990) Biochemistry, 29, 1255–1261.CrossRefPubMedGoogle Scholar
  28. 28.
    Nandi, P. K. (1998) Int. J. Biol. Macromol., 22, 23–31.CrossRefPubMedGoogle Scholar
  29. 29.
    Lala, A. K., and Kaul, P. (1992) J. Biol. Chem., 267, 19914–19918.PubMedGoogle Scholar
  30. 30.
    Fink, A. L., Calciano, C. T., Goto, Y., Kurotsu, T., and Palleros, D. R. (1994) Biochemistry, 33, 12504–125011.CrossRefPubMedGoogle Scholar
  31. 31.
    Wu, L. C., and Kim, P. S. (1998) J. Mol. Biol., 280, 175–182.CrossRefPubMedGoogle Scholar
  32. 32.
    Goto, Y., Takahashi, N., and Fink, A. L. (1990) Biochemistry, 29, 3480–3488.CrossRefPubMedGoogle Scholar
  33. 33.
    Hamada, D., Kuroda, Y., Kataoka, M., Aimoto, S., Yoshimura, T., and Goto, Y. (1996) J. Mol. Biol., 256, 172–186.CrossRefPubMedGoogle Scholar
  34. 34.
    Kataoka, M., Hagihara, Y., Mihara, K., and Goto, Y. (1993) J. Mol. Biol., 229, 591–596.CrossRefPubMedGoogle Scholar
  35. 35.
    Raschke, T. M., and Marqusee, S. (1997) Nat. Struct. Biol., 4, 298–304.CrossRefPubMedGoogle Scholar
  36. 36.
    Kay, M. S., and Baldwin, R. L. (1996) Nat. Struct. Biol., 3, 439–445.CrossRefPubMedGoogle Scholar
  37. 37.
    Khorasanizadeh, S., Peters, I. D., and Roder, H. (1996) Nat. Struct. Biol., 3, 193–205.CrossRefPubMedGoogle Scholar
  38. 38.
    Mitra, N., Srinivas, V. R., Ramya, T. N., Ahmad, N., Reddy, G. B., and Surolia, A. (2002) Biochemistry, 41, 9256–9263.CrossRefPubMedGoogle Scholar
  39. 39.
    Reddy, G. B., Srinivas, V. R., Ahmad, N., and Surolia, A. (1999) J. Biol. Chem., 274, 4500–4504.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  1. 1.Interdisciplinary Biotechnology UnitAligarh Muslim UniversityAligarhIndia
  2. 2.Department of Biochemistry, Life ScienceAMUAligarhIndia

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