Jump to ContentJump to Main Navigation
Show Summary Details
More options …


More options …
Volume 62, Issue 3


A stopped-flow fluorescence study of the native and modified lysozyme

Khosrow Khalifeh
  • Department of Biophysics & Biochemistry, Faculty of Sciences, Tarbiat Modares University, P.O. Box, 14115-175, Tehran, Islamic Republic of Iran
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Bijan Ranjbar
  • Department of Biophysics & Biochemistry, Faculty of Sciences, Tarbiat Modares University, P.O. Box, 14115-175, Tehran, Islamic Republic of Iran
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Khosro Khajeh
  • Department of Biophysics & Biochemistry, Faculty of Sciences, Tarbiat Modares University, P.O. Box, 14115-175, Tehran, Islamic Republic of Iran
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Hossein Naderi-Manesh
  • Department of Biophysics & Biochemistry, Faculty of Sciences, Tarbiat Modares University, P.O. Box, 14115-175, Tehran, Islamic Republic of Iran
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Mehdi Sadeghi / Sara Gharavi
Published Online: 2007-06-01 | DOI: https://doi.org/10.2478/s11756-007-0045-0


The protein folding kinetics of hen egg white lysozyme (HEWL) was studied using experimental and bioinformatics tools. The structure of the transition state in the unfolding pathway of lysozyme was determined with stopped-flow kinetics using intact HEWL and its chemically modified derivative, in which six lysine residues have been modified. The overall consistency of φ-value (φ ≈ 1) indicates that lysine side chains interactions are subject to breaking in the structure of the transition state. Following experimental evidences, multiple sequence alignment of lysozyme family in vertebrates and exact structural examination of lysozyme, showed that the α-helix in the structure of lysozyme has critical role in the unfolding kinetics.

Keywords: folding; stopped-flow kinetics; hen egg white lysozyme; φ-value; bioinformatics; transition state

  • [1] Altschul S.F., Gish W., Miller W., Myers E.W & Lipman D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403–410. Google Scholar

  • [2] Blake C.C.F., Johnson L.N., Mair G.A., North A.C.T., Phillips D.C & Sarma V.R. 1967. Crystallographic studies of the activity of hen egg-white lysozyme. Proc. Roy. Soc. 167: 378–388. Google Scholar

  • [3] Bryngelson J.D., Onuchic J.N., Socci N.D & Wolynes P.G. 1995. Funnels, pathways and the energy landscape of protein folding: a synthesis. Proteins: Struct. Funct. Genet. 21: 167–195. http://dx.doi.org/10.1002/prot.340210302CrossrefGoogle Scholar

  • [4] Caffotte A.F., Guillou Y & Goldberg M.E. 1992. Kinetic resolution of peptide bond and side-chain far UV CD during folding of HEWL. Biochemistry 31: 9694–9702. http://dx.doi.org/10.1021/bi00155a024CrossrefGoogle Scholar

  • [5] Chen L., Wildegger G., Kiefhaber T.H., Hodgson K.O & Doniach S. 1998. Kinetics of lysozyme refolding: structural characterization of a non-specifically collapsed state using time-resolved X-ray scattering. J. Mol. Biol. 276: 225–237. http://dx.doi.org/10.1006/jmbi.1997.1514CrossrefGoogle Scholar

  • [6] Dalby P.A, Oliveberg M. & Fersht A.R. 1998. Folding intermediates of wild-type and mutants of barnase: use of φ-value analysis and m-values to probe the cooperative nature of the folding pre-equilibrium. J. Mol. Biol. 276: 625–646. http://dx.doi.org/10.1006/jmbi.1997.1546CrossrefGoogle Scholar

  • [7] Demirel M.C., Atilgan A.R., Jernigan R.L., Erman B & Bahar I. 1998. Identification of kinetically hot residues in proteins. Protein Sci. 7: 2522–2532. PubMedCrossrefGoogle Scholar

  • [8] Denton M.E., Rothwarf D.M & Scheraga H.A. 1994. Kinetics of folding of guanidinic denatured hen egg white lysozyme and carboxymethyl Cys(6).Cys(12r)-lysozyme: a stopped-flow absorbance and fluorescence study. Biochemistry 33: 11225–11236. http://dx.doi.org/10.1021/bi00203a019CrossrefGoogle Scholar

  • [9] Dixon H.B.F & Perham R.N. 1968. Reversible blocking of amino groups with citraconic anhydride. Biochem. J. 109: 312–314. Google Scholar

  • [10] Evans M.G & Polanyi M. 1935. Some applications of the transition state method to the calculation of reaction velocities, especially in solution. Trans. Faraday Soc. 31: 875–894. http://dx.doi.org/10.1039/tf9353100875CrossrefGoogle Scholar

  • [11] Eyring H. 1935. The activated complex and the absolute rate of chemical reactions. Chem. Rev. 17: 65–77. http://dx.doi.org/10.1021/cr60056a006CrossrefGoogle Scholar

  • [12] Fersht A.R. 1993. Protein folding and stability: the pathway of folding of barnase. FEBS Lett. 325: 5–16. http://dx.doi.org/10.1016/0014-5793(93)81405-OCrossrefGoogle Scholar

  • [13] Fersht A.R. 1997. Nucleation mechanisms in protein folding. Curr. Opin. Struct. Biol. 7: 3–9. http://dx.doi.org/10.1016/S0959-440X(97)80002-4CrossrefGoogle Scholar

  • [14] Fersht A.R., Matouscheck A & Serrano L. 1992. The folding of an enzyme: I. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224: 771–782. http://dx.doi.org/10.1016/0022-2836(92)90561-WCrossrefGoogle Scholar

  • [15] Ikeguchi M., Fujino M., Kato M., Kuwajima K & Sugai S. 1998. Transition state in the folding of α-lactalbumin probed by the 6–120 disulfide bond. Protein Sci. 7: 1564–1574. http://dx.doi.org/10.1002/pro.5560070710CrossrefGoogle Scholar

  • [16] Imoto T., Forster L.S., Rupley J.A & Tanak F. 1981. Fluorescence of lysozyme: emission from tryptophan residues 62 and 108 and energy migration. Proc. Natl. Acad. Sci. USA 69: 1151–1155. http://dx.doi.org/10.1073/pnas.69.5.1151CrossrefGoogle Scholar

  • [17] Itzhaki L.S., Evans P.A., Dobson C.M. & Radford S.E. 1994. Tertiary interactions in the folding pathway of hen lysozyme: kinetic studies using fluorescent probs. Biochemistry 33: 5212–5220. http://dx.doi.org/10.1021/bi00183a026CrossrefGoogle Scholar

  • [18] Jolles P & Jolles J. 1984. What’s new in lysozyme research? Mol. Cell. Biochem. 63: 165–189. http://dx.doi.org/10.1007/BF00285225CrossrefGoogle Scholar

  • [19] Kato S., Shimoto N. & Utijma H. 1982. Identification and characterization of the direct folding process of hen egg white lysozyme. Biochemistry 21: 38–43. http://dx.doi.org/10.1021/bi00530a007CrossrefGoogle Scholar

  • [20] Khan F., Chuang J.I., Gianni S & Fersht A.R. 2003. The kinetic pathway of folding of barnase, J. Mol. Biol. 333: 169–186. http://dx.doi.org/10.1016/j.jmb.2003.08.024CrossrefGoogle Scholar

  • [21] Kiefhaber T. 1995. Kinetic traps in lysozyme folding. Proc. Natl. Acad. Sci. USA 92: 9029–9033. http://dx.doi.org/10.1073/pnas.92.20.9029CrossrefGoogle Scholar

  • [22] Kiefhaber T & Wildegger G. 1997. Three-state model for lysozyme folding: triangular folding mechanism with an energetically trapped intermediate. J. Mol. Biol. 270: 294–304. http://dx.doi.org/10.1006/jmbi.1997.1030CrossrefGoogle Scholar

  • [23] Kuwajima K., Hiraoka Y., Ikeguchi M & Sugai S. 1985. Comparison of the transition state folding intermediates in lysozyme and α-lactalbumin. Biochemistry 24: 874–881 http://dx.doi.org/10.1021/bi00325a010CrossrefGoogle Scholar

  • [24] Matouschek A., Kellis J., Serrano L & Fersht A.R. 1989. Mapping the transition state and pathway of protein folding by protein engineering. Nature 340: 122–126. http://dx.doi.org/10.1038/340122a0CrossrefGoogle Scholar

  • [25] Matouschek A., Serrano L. & Fersht A.R. 1992. The folding of an enzyme: IV. Structure of an intermediate in the refolding of barnase analysed by a protein engineering procedure. J. Mol. Biol. 224: 819–835. http://dx.doi.org/10.1016/0022-2836(92)90564-ZCrossrefGoogle Scholar

  • [26] Matouschek A., Serrano L., Meiering E.M., Bycroft M. & Fersht A.R. 1992. The folding of an enzyme: V. H/2H exchange-nuclear magnetic resonance studies on the folding pathway of barnase: complementarity to and agreement with protein engineering studies. J. Mol. Biol. 224: 837–845. http://dx.doi.org/10.1016/0022-2836(92)90565-2CrossrefGoogle Scholar

  • [27] Mirny L.A., Abkevich V.I & Shakhnovich E.I. 1998. How evolution makes proteins fold quickly. Proc. Natl. Acad. Sci. USA 95: 4976–4981. http://dx.doi.org/10.1073/pnas.95.9.4976CrossrefGoogle Scholar

  • [28] Mirny L.A & Shakhnovich E.I. 1999. Universally conserved positions in protein folds: reading evolutionary signals about stability, folding and function. J. Mol. Biol. 291: 177–196. http://dx.doi.org/10.1006/jmbi.1999.2911CrossrefGoogle Scholar

  • [29] Mirny L.A & Shakhnovich E.I. 2001a. Protein folding theory: from lattice to all-atom models. Annu. Rev. Biophys. Biomol. Struct. 30: 361–396. http://dx.doi.org/10.1146/annurev.biophys.30.1.361Google Scholar

  • [30] Mirny L.A & Shakhnovich E.I. 2001b. Evolutionary conservation of the folding nucleus. J. Mol. Biol. 308: 123–129. http://dx.doi.org/10.1006/jmbi.2001.4602Google Scholar

  • [31] O’Farrell P.H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250: 4007–4021. Google Scholar

  • [32] Pane V.S., Grosberg A.Y., Tanaka T & Rokhsar D.S. 1998. Pathways for protein folding: is a new view needed? Curr. Opin. Struct. Biol. 8: 68–79. http://dx.doi.org/10.1016/S0959-440X(98)80012-2CrossrefGoogle Scholar

  • [33] Parker M.J., Spencer J & Clark A.R. 1995. An integrated kinetic analysis of intermediates and transitions states in protein folding reactions. J. Mol. Biol. 253: 771–786. http://dx.doi.org/10.1006/jmbi.1995.0590CrossrefGoogle Scholar

  • [34] Plaxco K.W., Riddle D.S., Larson S., Ruczinski I., Thayer E.C & Buchwits B. 2001. Evolutionary conservation and protein folding kinetics. J. Mol. Biol. 298: 303–312. http://dx.doi.org/10.1006/jmbi.1999.3663CrossrefGoogle Scholar

  • [35] Poupon A. & Marnon J.P. 1999. Predicting the protein folding nucleus from a sequence. FEBS Lett. 452: 283–289. http://dx.doi.org/10.1016/S0014-5793(99)00622-5CrossrefGoogle Scholar

  • [36] Protasevich I., Ranjbar B., Lobachov V., Makarov A., Gilli R., Briand C., Lafitte D & Haiech J. 1997. Conformation and thermal denaturation of apocalmodulin: role of electrostatic mutations. Biochemistry 36: 2017–2024. http://dx.doi.org/10.1021/bi962538gCrossrefGoogle Scholar

  • [37] Radford S.E., Dobson C.M & Evans P.A. 1992. The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature 358: 302–307. http://dx.doi.org/10.1038/358302a0CrossrefGoogle Scholar

  • [38] Rypniech W.R., Holden H.M & Rayment I. 1993. Structural consequences of reductive methylation of lysine residue in hen egg white lysozyme: an X-ray analysis at 1.8–? resolution. Biochemistry 32: 9851–9858. http://dx.doi.org/10.1021/bi00088a041CrossrefGoogle Scholar

  • [39] Salmine M., Caro B., Guen-Robin F.L., Blais J.C & Jaouen G. 2004. Solution-and crystal-phase covalent modification of lysozyme by a purpose-designed organoruthenium complex. A MALDI-TOF MS study of its metal binding sites. ChemBioChem 5: 99–109. http://dx.doi.org/10.1002/cbic.200300637CrossrefGoogle Scholar

  • [40] Sanz J.M & Fersht A.R. 1994. Measurement of barnase refolding rate constants under denaturing conditions, FEBS Lett. 344: 216–220. http://dx.doi.org/10.1016/0014-5793(94)00384-XCrossrefGoogle Scholar

  • [41] Schippers P.H & Deckers H.P.J.M. 1981. Direct determination of absolute circular dichroism data and calibration of commercial instrument. Anal. Chem. 53: 778–788. http://dx.doi.org/10.1021/ac00229a008CrossrefGoogle Scholar

  • [42] Segel D.J., Bachmann A., Hofrichter J., Hodgson K.O & Daniach S. 1999. Characterization of transient intermediates in lysozyme folding with time-resolved small-angle X-ray scattering. J. Mol. Biol. 288: 489–499. http://dx.doi.org/10.1006/jmbi.1999.2703CrossrefGoogle Scholar

  • [43] Serrano L., Kellis J., Cann T.P., Matouschek A & Fersht A.R. 1992. The folding of an enzyme: II. Substructure of barnase and the contribution of different interactions to protein stability. J. Mol. Biol. 224: 783–804. http://dx.doi.org/10.1016/0022-2836(92)90562-XCrossrefGoogle Scholar

  • [44] Serrano L., Matouschek A & Fersht A.R. 1992. The folding of an enzyme: III. Structure of the transition state for unfolding of barnase analysed by a protein engineering procedure. J. Mol. Biol. 224: 805–818. http://dx.doi.org/10.1016/0022-2836(92)90563-YCrossrefGoogle Scholar

  • [45] Serrano L., Matouschek A & Fersht, A.R. 1992. The folding of an enzyme: IV. The folding pathway of barnase: comparison with theoretical models. J. Mol. Biol. 224: 847–850. http://dx.doi.org/10.1016/0022-2836(92)90566-3CrossrefGoogle Scholar

  • [46] Shakhnovich E.I., Abkevich V.I & Ptitsyn O. 1996. Conserved residues and the mechanism of protein folding. Nature 379: 96–98. http://dx.doi.org/10.1038/379096a0CrossrefGoogle Scholar

  • [47] Shrivastava I., Vishveshwara S., Clieplak M., Maritan A & Banavar J.R. 1995. Lattice model for rapidly folding protein-like heteropolymers. Proc. Natl. Acad. Sci. USA 92: 9206–9209. http://dx.doi.org/10.1073/pnas.92.20.9206CrossrefGoogle Scholar

  • [48] Suckau D., Mak M & Przybylski M. 1992. Protein surface topology-probing by selective chemical modification and mass spectrometric peptide mapping. Proc. Natl. Acad. Sci. USA 89: 5630–5634. http://dx.doi.org/10.1073/pnas.89.12.5630CrossrefGoogle Scholar

  • [49] Takakuwa T., Konno T & Meguro H.A. 1985. New standard substance for calibration of circular dichroism: ammoniumd-10-camphorsulfonate. Anal. Sci. 1: 215–225. Google Scholar

  • [50] Tanford C. 1968. Protein denaturation. Adv. Protein Chem. 23: 121–282. CrossrefGoogle Scholar

  • [51] Tanford C. 1970. Protein denaturation. Adv. Protein Chem. 24: 1–95. http://dx.doi.org/10.1016/S0065-3233(08)60241-7CrossrefGoogle Scholar

  • [52] Tanford C., Aune K.C & Ikai A.A. 1973. Kinetics of unfolding and refolding of proteins. III: Results for lysozyme. J. Mol. Biol. 73: 185–197. http://dx.doi.org/10.1016/0022-2836(73)90322-7CrossrefGoogle Scholar

  • [53] Tang K.S., Guaralnick B.J., Wang W.K., Fersht A.R & Itzhaki L.S. 1999. Stability and folding of the tumor suppressor protein P16. J. Mol. Biol. 285: 1869–1886. http://dx.doi.org/10.1006/jmbi.1998.2420CrossrefGoogle Scholar

  • [54] Thompson J.D., Higgins D.G & Gibson T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673–4680. http://dx.doi.org/10.1093/nar/22.22.4673CrossrefGoogle Scholar

About the article

Published Online: 2007-06-01

Published in Print: 2007-06-01

Citation Information: Biologia, Volume 62, Issue 3, Pages 258–264, ISSN (Online) 1336-9563, ISSN (Print) 0006-3088, DOI: https://doi.org/10.2478/s11756-007-0045-0.

Export Citation

© 2007 Institute of Molecular Biology, Slovak Academy of Sciences. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

Forough Hakiminia, Bijan Ranjbar, Khosrow Khalifeh, and khosro khajeh
International Journal of Biological Macromolecules, 2013, Volume 55, Page 123
Bijan Ranjbar and Pooria Gill
Chemical Biology & Drug Design, 2009, Volume 74, Number 2, Page 101

Comments (0)

Please log in or register to comment.
Log in