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. 1997 Jan 15;321(Pt 2):457–464. doi: 10.1042/bj3210457

Characterization of a partially structured state in an all-beta-sheet protein.

T Sivaraman 1, T K Kumar 1, G Jayaraman 1, C C Han 1, C Yu 1
PMCID: PMC1218091  PMID: 9020881

Abstract

Cardiotoxin analogue III (CTX III) is a low-molecular-mass all-beta-sheet protein isolated from the Taiwan cobra (Naja naja atra) venom. A stable partially structured state similar to the "molten globule' state has been identified for CTX III in a 3% (w/v) solution of 2,2,2-trichloroacetic acid at 298 K. This stable state has been structurally characterized using a variety of techniques such as CD, 1-anilinonaphthalene-8-sulphonate fluorescence binding, Fourier transform IR and two-dimensional NMR spectroscopy techniques. Direct assignment of the homonuclear two-dimensional NMR spectra of the protein in 3% trichloroacetic acid showed that drastic structural perturbation had not taken place in the protein and that the 'intermediate' state retained a significant portion of the native secondary-structural interactions. It is found that about 65% of the native beta-sheet structural contacts are maintained in the partially structured state of CTX III in 3% trichloroacetic acid.

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Selected References

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  1. Anfinsen C. B. Principles that govern the folding of protein chains. Science. 1973 Jul 20;181(4096):223–230. doi: 10.1126/science.181.4096.223. [DOI] [PubMed] [Google Scholar]
  2. Baldwin R. L. The nature of protein folding pathways: the classical versus the new view. J Biomol NMR. 1995 Feb;5(2):103–109. doi: 10.1007/BF00208801. [DOI] [PubMed] [Google Scholar]
  3. Barrick D., Baldwin R. L. Stein and Moore Award address. The molten globule intermediate of apomyoglobin and the process of protein folding. Protein Sci. 1993 Jun;2(6):869–876. doi: 10.1002/pro.5560020601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bhaskaran R., Huang C. C., Chang D. K., Yu C. Cardiotoxin III from the Taiwan cobra (Naja naja atra). Determination of structure in solution and comparison with short neurotoxins. J Mol Biol. 1994 Jan 28;235(4):1291–1301. doi: 10.1006/jmbi.1994.1082. [DOI] [PubMed] [Google Scholar]
  5. Buck M., Radford S. E., Dobson C. M. A partially folded state of hen egg white lysozyme in trifluoroethanol: structural characterization and implications for protein folding. Biochemistry. 1993 Jan 19;32(2):669–678. doi: 10.1021/bi00053a036. [DOI] [PubMed] [Google Scholar]
  6. Buck M., Schwalbe H., Dobson C. M. Characterization of conformational preferences in a partly folded protein by heteronuclear NMR spectroscopy: assignment and secondary structure analysis of hen egg-white lysozyme in trifluoroethanol. Biochemistry. 1995 Oct 10;34(40):13219–13232. doi: 10.1021/bi00040a038. [DOI] [PubMed] [Google Scholar]
  7. Bychkova V. E., Berni R., Rossi G. L., Kutyshenko V. P., Ptitsyn O. B. Retinol-binding protein is in the molten globule state at low pH. Biochemistry. 1992 Aug 25;31(33):7566–7571. doi: 10.1021/bi00148a018. [DOI] [PubMed] [Google Scholar]
  8. Bychkova V. E., Dujsekina A. E., Klenin S. I., Tiktopulo E. I., Uversky V. N., Ptitsyn O. B. Molten globule-like state of cytochrome c under conditions simulating those near the membrane surface. Biochemistry. 1996 May 14;35(19):6058–6063. doi: 10.1021/bi9522460. [DOI] [PubMed] [Google Scholar]
  9. Byler D. M., Susi H. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers. 1986 Mar;25(3):469–487. doi: 10.1002/bip.360250307. [DOI] [PubMed] [Google Scholar]
  10. Carlsson U., Jonsson B. H. Folding of beta-sheet proteins. Curr Opin Struct Biol. 1995 Aug;5(4):482–487. doi: 10.1016/0959-440x(95)80032-8. [DOI] [PubMed] [Google Scholar]
  11. Clark A. H., Saunderson D. H., Suggett A. Infrared and laser-Raman spectroscopic studies of thermally-induced globular protein gels. Int J Pept Protein Res. 1981 Mar;17(3):353–364. doi: 10.1111/j.1399-3011.1981.tb02002.x. [DOI] [PubMed] [Google Scholar]
  12. Dobson C. M., Evans P. A., Radford S. E. Understanding how proteins fold: the lysozyme story so far. Trends Biochem Sci. 1994 Jan;19(1):31–37. doi: 10.1016/0968-0004(94)90171-6. [DOI] [PubMed] [Google Scholar]
  13. Dobson C. M. Protein folding. Solid evidence for molten globules. Curr Biol. 1994 Jul 1;4(7):636–640. doi: 10.1016/s0960-9822(00)00141-x. [DOI] [PubMed] [Google Scholar]
  14. Englander S. W., Wand A. J. Main-chain-directed strategy for the assignment of 1H NMR spectra of proteins. Biochemistry. 1987 Sep 22;26(19):5953–5958. doi: 10.1021/bi00393a001. [DOI] [PubMed] [Google Scholar]
  15. Fan P., Bracken C., Baum J. Structural characterization of monellin in the alcohol-denatured state by NMR: evidence for beta-sheet to alpha-helix conversion. Biochemistry. 1993 Feb 16;32(6):1573–1582. doi: 10.1021/bi00057a023. [DOI] [PubMed] [Google Scholar]
  16. Fersht A. R. The sixth Datta Lecture. Protein folding and stability: the pathway of folding of barnase. FEBS Lett. 1993 Jun 28;325(1-2):5–16. doi: 10.1016/0014-5793(93)81405-o. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Goto Y., Nishikiori S. Role of electrostatic repulsion in the acidic molten globule of cytochrome c. J Mol Biol. 1991 Dec 5;222(3):679–686. doi: 10.1016/0022-2836(91)90504-y. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Harding M. M., Williams D. H., Woolfson D. N. Characterization of a partially denatured state of a protein by two-dimensional NMR: reduction of the hydrophobic interactions in ubiquitin. Biochemistry. 1991 Mar 26;30(12):3120–3128. doi: 10.1021/bi00226a020. [DOI] [PubMed] [Google Scholar]
  21. Hughson F. M., Wright P. E., Baldwin R. L. Structural characterization of a partly folded apomyoglobin intermediate. Science. 1990 Sep 28;249(4976):1544–1548. doi: 10.1126/science.2218495. [DOI] [PubMed] [Google Scholar]
  22. Jagannadham M. V., Balasubramanian D. The molten globular intermediate form in the folding pathway of human carbonic anhydrase B. FEBS Lett. 1985 Sep 2;188(2):326–330. doi: 10.1016/0014-5793(85)80396-3. [DOI] [PubMed] [Google Scholar]
  23. Jeng M. F., Englander S. W. Stable submolecular folding units in a non-compact form of cytochrome c. J Mol Biol. 1991 Oct 5;221(3):1045–1061. doi: 10.1016/0022-2836(91)80191-v. [DOI] [PubMed] [Google Scholar]
  24. Jennings P. A., Wright P. E. Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science. 1993 Nov 5;262(5135):892–896. doi: 10.1126/science.8235610. [DOI] [PubMed] [Google Scholar]
  25. Kamatari Y. O., Konno T., Kataoka M., Akasaka K. The methanol-induced globular and expanded denatured states of cytochrome c: a study by CD fluorescence, NMR and small-angle X-ray scattering. J Mol Biol. 1996 Jun 14;259(3):512–523. doi: 10.1006/jmbi.1996.0336. [DOI] [PubMed] [Google Scholar]
  26. Kumar T. K., Jayaraman G., Lee C. S., Sivaraman T., Lin W. Y., Yu C. Identification of 'molten globule'-like state in all beta-sheet protein. Biochem Biophys Res Commun. 1995 Feb 15;207(2):536–543. doi: 10.1006/bbrc.1995.1221. [DOI] [PubMed] [Google Scholar]
  27. Kumar T. K., Lee C. S., Yu C. A case study of cardiotoxin III from the Taiwan cobra (Naja naja atra). Solution structure and other physical properties. Adv Exp Med Biol. 1996;391:115–129. doi: 10.1007/978-1-4613-0361-9_7. [DOI] [PubMed] [Google Scholar]
  28. Kumar T. K., Subbiah V., Ramakrishna T., Pandit M. W. Trichloroacetic acid-induced unfolding of bovine pancreatic ribonuclease. Existence of molten globule-like state. J Biol Chem. 1994 Apr 29;269(17):12620–12625. [PubMed] [Google Scholar]
  29. Kuwajima K. The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins. 1989;6(2):87–103. doi: 10.1002/prot.340060202. [DOI] [PubMed] [Google Scholar]
  30. Liu Z. P., Rizo J., Gierasch L. M. Equilibrium folding studies of cellular retinoic acid binding protein, a predominantly beta-sheet protein. Biochemistry. 1994 Jan 11;33(1):134–142. doi: 10.1021/bi00167a017. [DOI] [PubMed] [Google Scholar]
  31. Miranker A., Robinson C. V., Radford S. E., Aplin R. T., Dobson C. M. Detection of transient protein folding populations by mass spectrometry. Science. 1993 Nov 5;262(5135):896–900. doi: 10.1126/science.8235611. [DOI] [PubMed] [Google Scholar]
  32. Otzen D. E., Itzhaki L. S., elMasry N. F., Jackson S. E., Fersht A. R. Structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10422–10425. doi: 10.1073/pnas.91.22.10422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ptitsyn O. B. Molten globule and protein folding. Adv Protein Chem. 1995;47:83–229. doi: 10.1016/s0065-3233(08)60546-x. [DOI] [PubMed] [Google Scholar]
  34. Sagar A. J., Pandit M. W. Denaturation studies on bovine pancreatic ribonuclease. Effect of trichloroacetic acid. Biochim Biophys Acta. 1983 Mar 30;743(3):303–309. doi: 10.1016/0167-4838(83)90386-2. [DOI] [PubMed] [Google Scholar]
  35. Scholtz J. M., Baldwin R. L. Perchlorate-induced denaturation of ribonuclease A: investigation of possible folding intermediates. Biochemistry. 1993 May 4;32(17):4604–4608. doi: 10.1021/bi00068a017. [DOI] [PubMed] [Google Scholar]
  36. Sosnick T. R., Mayne L., Hiller R., Englander S. W. The barriers in protein folding. Nat Struct Biol. 1994 Mar;1(3):149–156. doi: 10.1038/nsb0394-149. [DOI] [PubMed] [Google Scholar]
  37. Surewicz W. K., Mantsch H. H., Chapman D. Determination of protein secondary structure by Fourier transform infrared spectroscopy: a critical assessment. Biochemistry. 1993 Jan 19;32(2):389–394. doi: 10.1021/bi00053a001. [DOI] [PubMed] [Google Scholar]
  38. Yang C. C., King K., Sun T. P. Chemical modification of lysine and histidine residues in phospholipase A2 from the venom of Naja naja atra (Taiwan cobra). Toxicon. 1981;19(5):645–659. doi: 10.1016/0041-0101(81)90102-1. [DOI] [PubMed] [Google Scholar]

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