Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Jan;8(1):35–44. doi: 10.1110/ps.8.1.35

A near-native state on the slow refolding pathway of hen lysozyme.

S K Kulkarni 1, A E Ashcroft 1, M Carey 1, D Masselos 1, C V Robinson 1, S E Radford 1
PMCID: PMC2144118  PMID: 10210181

Abstract

The refolding of four disulfide lysozyme (at pH 5.2, 20 degrees C) involves parallel pathways, which have been proposed to merge at a near-native state. This species contains stable structure in the alpha- and beta-domains but lacks a functional active site. Although previous experiments have demonstrated that the near-native state is populated on the fast refolding pathway, its relevance to slow refolding molecules could not be directly determined from previous experiments. In this paper, we describe experiments that investigate the effect of added salts on the refolding pathway of lysozyme at pH 5.2, 20 degrees C. We show, using stopped flow tryptophan fluorescence, inhibitor binding, and circular dichroism (CD), that the rate of formation of native lysozyme on the slow refolding track is significantly reduced in solutions of high ionic strength in a manner dependent on the position of the anion in the Hofmeister series. By contrast, the rate of evolution of hydrogen exchange (HX) protection monitored by electrospray ionization mass spectrometry (ESI MS) is unchanged under the refolding conditions studied. The data show, therefore, that at high ionic strengths beta-domain stabilization and native state formation on the slow refolding pathway become kinetically decoupled such that the near-native state becomes significantly populated. Thus, by changing the energy landscape with the addition of salts new insights into the relevance of intermediate states in lysozyme refolding are revealed.

Full Text

The Full Text of this article is available as a PDF (675.6 KB).

Selected References

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

  1. Bai Y., Milne J. S., Mayne L., Englander S. W. Primary structure effects on peptide group hydrogen exchange. Proteins. 1993 Sep;17(1):75–86. doi: 10.1002/prot.340170110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baldwin R. L. How Hofmeister ion interactions affect protein stability. Biophys J. 1996 Oct;71(4):2056–2063. doi: 10.1016/S0006-3495(96)79404-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blake C. C., Koenig D. F., Mair G. A., North A. C., Phillips D. C., Sarma V. R. Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Angstrom resolution. Nature. 1965 May 22;206(4986):757–761. doi: 10.1038/206757a0. [DOI] [PubMed] [Google Scholar]
  4. Booth D. R., Sunde M., Bellotti V., Robinson C. V., Hutchinson W. L., Fraser P. E., Hawkins P. N., Dobson C. M., Radford S. E., Blake C. C. Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature. 1997 Feb 27;385(6619):787–793. doi: 10.1038/385787a0. [DOI] [PubMed] [Google Scholar]
  5. Chaffotte A. F., Guillou Y., Goldberg M. E. Kinetic resolution of peptide bond and side chain far-UV circular dichroism during the folding of hen egg white lysozyme. Biochemistry. 1992 Oct 13;31(40):9694–9702. doi: 10.1021/bi00155a024. [DOI] [PubMed] [Google Scholar]
  6. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dill K. A., Chan H. S. From Levinthal to pathways to funnels. Nat Struct Biol. 1997 Jan;4(1):10–19. doi: 10.1038/nsb0197-10. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Gladwin S. T., Evans P. A. Structure of very early protein folding intermediates: new insights through a variant of hydrogen exchange labelling. Fold Des. 1996;1(6):407–417. doi: 10.1016/S1359-0278(96)00057-0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Itzhaki L. S., Evans P. A., Dobson C. M., Radford S. E. Tertiary interactions in the folding pathway of hen lysozyme: kinetic studies using fluorescent probes. Biochemistry. 1994 May 3;33(17):5212–5220. doi: 10.1021/bi00183a026. [DOI] [PubMed] [Google Scholar]
  14. Itzhaki L. S., Evans P. A. Solvent isotope effects on the refolding kinetics of hen egg-white lysozyme. Protein Sci. 1996 Jan;5(1):140–146. doi: 10.1002/pro.5560050117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jollès P., Jollès J. What's new in lysozyme research? Always a model system, today as yesterday. Mol Cell Biochem. 1984 Sep;63(2):165–189. doi: 10.1007/BF00285225. [DOI] [PubMed] [Google Scholar]
  16. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  17. Kato S., Okamura M., Shimamoto N., Utiyama H. Spectral evidence for a rapidly formed structural intermediate in the refolding kinetics of hen egg-white lysozyme. Biochemistry. 1981 Mar 3;20(5):1080–1085. doi: 10.1021/bi00508a006. [DOI] [PubMed] [Google Scholar]
  18. Kato S., Shimamoto N., Utiyama H. Identification and characterization of the direct folding process of hen egg-white lysozyme. Biochemistry. 1982 Jan 5;21(1):38–43. doi: 10.1021/bi00530a007. [DOI] [PubMed] [Google Scholar]
  19. Kiefhaber T. Kinetic traps in lysozyme folding. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9029–9033. doi: 10.1073/pnas.92.20.9029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lazaridis T., Karplus M. "New view" of protein folding reconciled with the old through multiple unfolding simulations. Science. 1997 Dec 12;278(5345):1928–1931. doi: 10.1126/science.278.5345.1928. [DOI] [PubMed] [Google Scholar]
  21. Matagne A., Chung E. W., Ball L. J., Radford S. E., Robinson C. V., Dobson C. M. The origin of the alpha-domain intermediate in the folding of hen lysozyme. J Mol Biol. 1998 Apr 17;277(5):997–1005. doi: 10.1006/jmbi.1998.1657. [DOI] [PubMed] [Google Scholar]
  22. Matagne A., Radford S. E., Dobson C. M. Fast and slow tracks in lysozyme folding: insight into the role of domains in the folding process. J Mol Biol. 1997 Apr 18;267(5):1068–1074. doi: 10.1006/jmbi.1997.0963. [DOI] [PubMed] [Google Scholar]
  23. McCrary B. S., Bedell J., Edmondson S. P., Shriver J. W. Linkage of protonation and anion binding to the folding of Sac7d. J Mol Biol. 1998 Feb 13;276(1):203–224. doi: 10.1006/jmbi.1998.1500. [DOI] [PubMed] [Google Scholar]
  24. McKenzie H. A., White F. H., Jr Lysozyme and alpha-lactalbumin: structure, function, and interrelationships. Adv Protein Chem. 1991;41:173–315. doi: 10.1016/s0065-3233(08)60198-9. [DOI] [PubMed] [Google Scholar]
  25. Miranker A., Radford S. E., Karplus M., Dobson C. M. Demonstration by NMR of folding domains in lysozyme. Nature. 1991 Feb 14;349(6310):633–636. doi: 10.1038/349633a0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Miranker A., Robinson C. V., Radford S. E., Dobson C. M. Investigation of protein folding by mass spectrometry. FASEB J. 1996 Jan;10(1):93–101. doi: 10.1096/fasebj.10.1.8566553. [DOI] [PubMed] [Google Scholar]
  28. Morgan C. J., Miranker A., Dobson C. M. Characterization of collapsed states in the early stages of the refolding of hen lysozyme. Biochemistry. 1998 Jun 9;37(23):8473–8480. doi: 10.1021/bi9731504. [DOI] [PubMed] [Google Scholar]
  29. Moult J., Yonath A., Traub W., Smilansky A., Podjarny A., Rabinovich D., Saya A. The structure of triclinic lysozyme at 2-5 A resolution. J Mol Biol. 1976 Jan 15;100(2):179–195. doi: 10.1016/s0022-2836(76)80147-7. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Pepys M. B., Hawkins P. N., Booth D. R., Vigushin D. M., Tennent G. A., Soutar A. K., Totty N., Nguyen O., Blake C. C., Terry C. J. Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature. 1993 Apr 8;362(6420):553–557. doi: 10.1038/362553a0. [DOI] [PubMed] [Google Scholar]
  32. Radford S. E., Dobson C. M., Evans P. A. The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature. 1992 Jul 23;358(6384):302–307. doi: 10.1038/358302a0. [DOI] [PubMed] [Google Scholar]
  33. Radford S. E., Dobson C. M. Insights into protein folding using physical techniques: studies of lysozyme and alpha-lactalbumin. Philos Trans R Soc Lond B Biol Sci. 1995 Apr 29;348(1323):17–25. doi: 10.1098/rstb.1995.0041. [DOI] [PubMed] [Google Scholar]
  34. Rothwarf D. M., Scheraga H. A. Role of non-native aromatic and hydrophobic interactions in the folding of hen egg white lysozyme. Biochemistry. 1996 Oct 29;35(43):13797–13807. doi: 10.1021/bi9608119. [DOI] [PubMed] [Google Scholar]
  35. Shih P., Holland D. R., Kirsch J. F. Thermal stability determinants of chicken egg-white lysozyme core mutants: hydrophobicity, packing volume, and conserved buried water molecules. Protein Sci. 1995 Oct;4(10):2050–2062. doi: 10.1002/pro.5560041010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sun D. P., Liao D. I., Remington S. J. Electrostatic fields in the active sites of lysozymes. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5361–5365. doi: 10.1073/pnas.86.14.5361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Timasheff S. N. The control of protein stability and association by weak interactions with water: how do solvents affect these processes? Annu Rev Biophys Biomol Struct. 1993;22:67–97. doi: 10.1146/annurev.bb.22.060193.000435. [DOI] [PubMed] [Google Scholar]
  38. Washabaugh M. W., Collins K. D. The systematic characterization by aqueous column chromatography of solutes which affect protein stability. J Biol Chem. 1986 Sep 25;261(27):12477–12485. [PubMed] [Google Scholar]
  39. Wildegger G., Kiefhaber T. Three-state model for lysozyme folding: triangular folding mechanism with an energetically trapped intermediate. J Mol Biol. 1997 Jul 11;270(2):294–304. doi: 10.1006/jmbi.1997.1030. [DOI] [PubMed] [Google Scholar]
  40. Williams M. A., Goodfellow J. M., Thornton J. M. Buried waters and internal cavities in monomeric proteins. Protein Sci. 1994 Aug;3(8):1224–1235. doi: 10.1002/pro.5560030808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wolynes P., Luthey-Schulten Z., Onuchic J. Fast-folding experiments and the topography of protein folding energy landscapes. Chem Biol. 1996 Jun;3(6):425–432. doi: 10.1016/s1074-5521(96)90090-3. [DOI] [PubMed] [Google Scholar]
  42. Yang J. J., Buck M., Pitkeathly M., Kotik M., Haynie D. T., Dobson C. M., Radford S. E. Conformational properties of four peptides spanning the sequence of hen lysozyme. J Mol Biol. 1995 Sep 29;252(4):483–491. doi: 10.1006/jmbi.1995.0513. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES