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Structural Analysis of Hen Egg Lysozyme Refolded after Denaturation at Acidic pH

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Abstract

Protein structures fluctuate in solution; therefore, proteins have multiple stable structures that are slightly different from each other. In this study, we determined the crystal structure of hen egg lysozyme refolded after denaturation at acidic pH (rHEL) and found a structure different from native HEL (nHEL). The different local conformations of the peptide bond between Asp101 and Gly102 found in the crystal structure was supported by the NMR results for nHEL and rHEL. The NMR experiments also showed shifts in the heteronuclear single quantum coherence signals derived from Thr43 and Asp52. The chemical shift change of Asp52 could be explained by the crystal structure of rHEL, showing the conformational change of Tyr53, whose phenol ring directly lies on the main chain of Asp52. The catalytic activity of rHEL was similar to that of nHEL, indicating that the conformational change had little effect on activity. In contrast, conformational changes could be detected by the binding of monoclonal antibodies against HEL. Using multiple methods, we successfully detected the unusual structure of HEL, which might be another stable structure of HEL in solution.

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References

  1. Dill KA, Chan HS (1997) From Levinthal to pathways to funnels. Nature Struct Biol 14:10–19. https://doi.org/10.1038/nsb0197-10

    Article  Google Scholar 

  2. Okazaki K, Takada S (2008) Dynamic energy landscape view of coupled binding and protein conformational change: induced-fit versus population-shift mechanisms. Proc Natl Acad Sci USA 105:11182–11187. https://doi.org/10.1073/pnas.0802524105

    Article  PubMed  PubMed Central  Google Scholar 

  3. Yao S, Lee EF, Pettikiriarachchi A, Evangelista M, Keizer DW, Fairlie WD (2016) Characterisation of the conformational preference and dynamics of the intrinsically disordered N-terminal region of Beclin 1 by NMR spectroscopy. Biochim Biophys Acta 1864:1128–1137. https://doi.org/10.1016/j.bbapap.2016.06.005

    Article  CAS  PubMed  Google Scholar 

  4. Kulkarni P, Solomon TL, He Y, Chen Y, Bryan PN, Orban J (2018) Structural metamorphism and polymorphism in proteins on the brink of thermodynamic stability. Protein Sci 27:1557–1567. https://doi.org/10.1002/pro.3458

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Blake CCF, Fenn RH, North ACT, Phillips DC, Poljak RJ (1962) Structure of lysozyme: a Fourier map of the electron density at 6 Å resolution obtained by X-ray diffraction. Nature 196:1173–1176. https://doi.org/10.1038/1961173a0

    Article  CAS  PubMed  Google Scholar 

  6. Phillips DC (1967) The hen egg white lysozyme molecule. Proc Natl Acad Sci USA 57:483–495. https://doi.org/10.1073/pnas.57.3.483

    Article  PubMed Central  Google Scholar 

  7. Strynadka NC, James MN (1991) Lysozyme revisited: crystallographic evidence for distortion of an N-acetylmuramic acid residue bound in site D. J Mol Biol 220:401–424. https://doi.org/10.1016/0022-2836(91)90021-w

    Article  CAS  PubMed  Google Scholar 

  8. Oda M, Kitai A, Murakami A, Nishimura M, Ohkuri T, Abe Y, Ueda T, Nakamura H, Azuma T (2010) Evaluation of the conformational equilibrium of reduced hen egg lysozyme by antibodies to the native form. Arch Biochem Biophys 494:145–150. https://doi.org/10.1016/j.abb.2009.11.024

    Article  CAS  PubMed  Google Scholar 

  9. Radford SE, Woolfson DN, Martin SR, Lowe G, Dobson CM (1991) A three-disulphide derivative of hen lysozyme. Structure, dynamics and stability. Biochem J 273:211–217. https://doi.org/10.1042/bj2730211

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Buck M, Boyd J, Redfield C, MacKenzie DA, Jeenes DJ, Archer DB, Dobson CM (1995) Structural determinants of protein dynamics: analysis of 15N NMR relaxation measurements for main-chain and side-chain nuclei of hen egg white lysozyme. Biochemistry 34:4041–4055. https://doi.org/10.1021/bi00012a023

    Article  CAS  PubMed  Google Scholar 

  11. Tomizawa H, Yamada H, Hashimoto Y, Imoto T (1995) Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences. Protein Eng 8:1023–1028. https://doi.org/10.1021/bi00195a019

    Article  CAS  PubMed  Google Scholar 

  12. Kamatari YO, Konno T, Kataoka M, Akasaka K (1998) The methanol-induced transition and the expanded helical conformation in hen lysozyme. Protein Sci 7:681–688. https://doi.org/10.1002/pro.5560070317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Klein-Seetharaman J, Oikawa M, Grimshaw SB, Wirmer J, Duchardt E, Ueda T, Imoto T, Smith LJ, Dobson CM, Schwalbe H (2002) Long-range interactions within a nonnative protein. Science 295:1719–1722. https://doi.org/10.1126/science.1067680

    Article  CAS  PubMed  Google Scholar 

  14. Thoppil AA, Kishore N (2007) Equimolar mixture of 2,2,2-trifluoroethanol and 4-chloro-1-butanol is a stronger inducer of molten globule state: isothermal titration calorimetric and spectroscopic studies. Protein J 26:507–516. https://doi.org/10.1007/s10930-007-9092-1

    Article  CAS  PubMed  Google Scholar 

  15. Cooper A, Eyles SJ, Radford SE, Dobson CM (1992) Thermodynamic consequences of the removal of a disulphide bridge from hen lysozyme. J Mol Biol. https://doi.org/10.1016/0022-2836(92)90094-Z

    Article  PubMed  Google Scholar 

  16. Oda M, Uchiyama S, Robinson CV, Fukui K, Kobayashi Y, Azuma T (2006) Regional and segmental flexibility of antibodies in interaction with antigens of different size. FEBS J 273:1476–1487. https://doi.org/10.1111/j.1742-4658.2006.05168.x

    Article  CAS  PubMed  Google Scholar 

  17. Liebschner D, Afonine PV, Baker ML, Bunkóczi G, Chen VB, Croll TI, Hintze B, Hung LW, Jain S, McCoy AJ, Moriarty NW, Oeffner RD, Poon BK, Prisant MG, Read RJ, Richardson JS, Richardson DC, Sammito MD, Sobolev OV, Stockwell DH, Terwilliger TC, Urzhumtsev AG, Videau LL, Williams CJ, Adams PD (2019) Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Cryst D75:861–877. https://doi.org/10.1107/S2059798319011471

    Article  Google Scholar 

  18. Brunger AT, Adams PD, Clore GM, Gros P, Grosse-Kunstleve RW, Jiang J-S, Kuszewski J, Nilges N, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography & NMR System (CNS), A new software suite for macromolecular structure determination. Acta Cryst D54:905–921. https://doi.org/10.1107/S0907444998003254

    Article  CAS  Google Scholar 

  19. Buck M, Radford SE, Dobson CM (1994) Amide hydrogen exchange in a highly denatured state. Hen egg-white lysozyme in urea. J Mol Biol 237:247–254. https://doi.org/10.1006/jmbi.1994.1228

    Article  CAS  PubMed  Google Scholar 

  20. Kamatari YO, Yamada H, Akasaka K, Jones JA, Dobson CM, Smith LJ (2001) Response of native and denatured hen lysozyme to high pressure studied by 15N/1H NMR spectroscopy. Eur J Biochem 268:1782–1793. https://doi.org/10.1046/j.1432-1327.2001.02050.x

    Article  CAS  PubMed  Google Scholar 

  21. Tomizawa H, Yamada H, Ueda T, Imoto T (1994) Isolation and characterization of 101-succinimide lysozyme that possesses the cyclic imide at Asp101-Gly102. Biochemistry 33:8770–8774. https://doi.org/10.1021/bi00195a019

    Article  CAS  PubMed  Google Scholar 

  22. Harata K (1994) X-ray structure of a monoclinic form of hen egg-white lysozyme crystallized at 313 K. Comparison of two independent molecules. Acta Cryst D50:250–257. https://doi.org/10.1107/S0907444993013290

    Article  CAS  Google Scholar 

  23. Noguchi S, Miyawaki K, Satow Y (1998) Succinimide and isoaspartate residues in the crystal structures of hen egg-white lysozyme complexed with tri-N-acetylchitotriose. J Mol Biol 278:231–238. https://doi.org/10.1006/jmbi.1998.1674

    Article  CAS  PubMed  Google Scholar 

  24. Masumoto K, Ueda T, Motoshima H, Imoto T (2000) Relationship between local structure and stability in hen egg white lysozyme mutant with alanine substituted for glycine. Protein Eng 13:691–695. https://doi.org/10.1093/protein/13.10.691

    Article  CAS  PubMed  Google Scholar 

  25. Baker D, Sohl JL, Agard DA (1992) A protein-folding reaction under kinetic control. Nature 356:263–265. https://doi.org/10.1038/356263a0

    Article  CAS  PubMed  Google Scholar 

  26. Eder J, Rheinnecker M, Fersht AR (1993) Folding of subtilisin BPN’: role of the pro-sequence. J Mol Biol 233:293–304. https://doi.org/10.1006/jmbi.1993.1507

    Article  CAS  PubMed  Google Scholar 

  27. Dee DR, Yada RY (2010) The prosegment catalyzes pepsin folding to a kinetically trapped native state. Biochemistry 49:365–371. https://doi.org/10.1021/bi9014055

    Article  CAS  PubMed  Google Scholar 

  28. Konno T, Kamatari YO, Tanaka N, Kamikubo H, Dobson CM, Nagayama K (2000) A partially unfolded structure of the alkaline-denatured state of pepsin and its implication for stability of the zymogen-derived protein. Biochemistry 39:4182–4190. https://doi.org/10.1021/bi991923d

    Article  CAS  PubMed  Google Scholar 

  29. Furukawa K, Oda M, Nakamura H (1996) A small engineered protein lacks structural uniqueness by increasing the side-chain conformational entropy. Proc Natl Acad Sci USA 93:13583–13588. https://doi.org/10.1073/pnas.93.24.13583

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Inaba S, Fukada H, Ikegami T, Oda M (2013) Thermodynamic effects of multiple protein conformations on stability and DNA binding. Arch Biochem Biophys 537:225–232. https://doi.org/10.1016/j.abb.2013.07.014

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank to Drs Akemi Ota and Takachika Azuma for providing hybridomas producing HyC1 and HyC2, and Ms Yumi Kitagawa for technical support. This study was supported by Nanken-Kyoten, Tokyo Medical and Dental University.

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Author notes

  1. Teikichi Ikura

    Present address: Institute for Human Life Innovation, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo, 112-8610, Japan

Authors and Affiliations

  1. Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, 606-8522, Japan

    Masayuki Oda & Tomoki Sano

  2. Institute for Glyco-core Research (iGCORE), Gifu University, 1-1 Yanagido, Gifu, 501-1194, Japan

    Yuji O. Kamatari

  3. School of Pharmacy, International University of Health and Welfare, 137-1 Enokizu, Okawa, Fukuoka, 831-8501, Japan

    Yoshito Abe

  4. Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan

    Teikichi Ikura & Nobutoshi Ito

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  1. Masayuki Oda
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Correspondence to Masayuki Oda or Nobutoshi Ito.

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Oda, M., Sano, T., Kamatari, Y.O. et al. Structural Analysis of Hen Egg Lysozyme Refolded after Denaturation at Acidic pH. Protein J 41, 71–78 (2022). https://doi.org/10.1007/s10930-021-10036-3

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