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Irreversible Nature of Mesoscopic Aggregates in Lysozyme Solutions


Study of protein stability in solutions is important for better understanding the pathogenesis of diseases caused by abnormal protein folding and aggregation. Lysozyme is known to form mesoscopic aggregates (30–100 nm radius) in concentrated solutions (>30 mg/mL), however the origin and thermodynamic status of these aggregates remain unclear. In this work we have investigated the effects of concentration, filtration, and temperature on the sizes and relative amount of mesoscale aggregates in solutions of lysozyme. We have used dynamic light scattering, small-angle X-ray scattering, and size exclusion chromatography (SEC). Mesoscopic protein aggregates were commonly thought to be in equilibrium with protein monomers in solution, resulting from a reversible self-assembly of the monomers. We instead show that systematic filtration through 20 nm pore size filters completely removes the aggregates from solution. The aggregates do not reemerge. Without filtering, the relative number of monomers decreases with increasing solution temperature, indicating formation of more aggregates. SEC was used to search for the presence of lysozyme dimers, which have been previously hypothesized to be related to the formation of mesoscopic aggregates. SEC did not detect dimers in solutions of filtered or unfiltered lysozyme. Taken together, our results strongly suggest that the mesoscopic aggregates in lysozyme are not caused by reversible self-assembly of lysozyme monomers and are not an intrinsic property of lysozyme monomers in their native state. We hypothesize that the lysozyme aggregation is likely due to some impurities in lysozyme introduced during purification or lyophilization and/or to traces of misfolded lysozyme.

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  1. Alam, P., Siddiqi, K., Chturvedi, S.K., and Khan, R.H., Int. J. Biol. Macromol., 2017, vol. 103, p. 208.

    CAS  Article  Google Scholar 

  2. Pan, W., Vekilov, P.G., and Lubchenko, V., J. Phys. Chem. B, 2010, vol. 114, p. 7620.

    CAS  Article  Google Scholar 

  3. Li, Y., Lubchenko, V., Vorontsova, M.A., Filobelo, L., and Vekilov, P.G., J. Phys. Chem. B, 2012, vol. 116, p. 10657.

    CAS  Article  Google Scholar 

  4. Vorontsova, M.A., Chan, H.Y., Lubchenko, V., and Vekilov, P.G., Biophys. J., 2015, vol. 109, p. 1959.

    CAS  Article  Google Scholar 

  5. Safari, M.S., Byington, M.C., Conrad, J.C., and Vekilov, P.G., J. Phys. Chem. B, 2017, vol. 121, p. 9091.

    CAS  Article  Google Scholar 

  6. Byington, M.C., Safari, M.S., Lubchenko, V., McCabe, J.W., Angel, L.A., Hawke, D.H., Bark, S.J., Conrad, J.C., and Vekilov, P.G., Weakly-Bound Dimers That Underlie the Crystal Nucleation Precursors in Lysozyme Solutions, bioRxiv, 2018, p. 275 222.

  7. Berne, B.J. and Pecora, R., Dynamic Light Scattering: with Applications to Chemistry, Biology, and Physics, New York: Wiley, 2000.

    Google Scholar 

  8. Van Holde, K.E., Johnson, W.C., and Ho, P.S., Principles of Physical Biochemistry, Upper Saddle River: Pearson Education, 2006.

    Google Scholar 

  9. Russel, W.B., Saville, D.A., and Schowalter, W.R., Colloidal Dispersions, New York: Cambridge Univ. Press, 1991.

    Google Scholar 

  10. Boldon, L., Laliberte, F., and Liu, L., Nano Rev., 2015, vol. 6, p. 25661.

    Article  Google Scholar 

  11. Guinier, A., Ann. Phys., 1939, vol. 11, no. 12, p. 161.

    Article  Google Scholar 

  12. Zheng, X., Anisimov, M.A., Sengers, J.V., and He, M., J. Phys. Chem. B, 2018, vol. 122, p. 3454.

    CAS  Article  Google Scholar 

  13. Parmar, A.S. and Muschol, M., Biophys. J., 2003, vol. 97, p. 590.

    Article  Google Scholar 

  14. Tande, B.M., Wagner, N.J., Mackay, M.E., Hawker, C.J., and Jeong, M., Macromolecules, 2001, vol. 34, p. 8580.

    CAS  Article  Google Scholar 

  15. Zimm, B.H., J. Chem. Phys., 1948, vol. 16, p. 1093.

    CAS  Article  Google Scholar 

  16. Zamora, P.C. and Zukoski, C.F., Langmuir, 1996, vol. 12, p. 3541.

    CAS  Article  Google Scholar 

  17. Davis, H.T. and Winkelmann, J., Statistical Mechanics of Phases, Interfaces, and Thin Films, New York: Wiley-VCH, 1996.

    Google Scholar 

  18. Saunders, A.E. and Korgel, B.A., J. Phys. Chem. B, 2004, vol. 108, p. 16732.

    CAS  Article  Google Scholar 

  19. Wang, W. and Roberts, C.J., Int. J. Pharm., 2018, vol. 550, p. 251.

    CAS  Article  Google Scholar 

  20. Thomas, B.R., Vekilov, P.G., and Rosenberger, F., Acta Crystallogr. D, 1996, vol. 52, p. 776.

    CAS  Article  Google Scholar 

  21. Parmar, A.S., Gottschall, P.E., and Muschol, M., Biophys. Chem., 2007, vol. 129, p. 224.

    CAS  Article  Google Scholar 

  22. Shchukin, E.D., Pertsov, A.V., Amelina, E.A., and Zelenev, A.S., Colloid and Surface Chemistry, Amsterdam: Elsevier, 2001.

    Google Scholar 

  23. Subramanian, D., Klauda, J.B., Collings, P.J., and Anisimov, M.A., J. Phys. Chem. B, 2014, vol. 118, p. 5994.

    CAS  Article  Google Scholar 

  24. Rak, D. and Sedlak, M., J. Phys. Chem. B, 2019, vol. 123, p. 1365.

    CAS  Article  Google Scholar 

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We thank Prof. Peter Vekilov for consulting us on the issue of mesoscopic aggregation in lysozyme solutions and Prof. Marc Taraban for advice on Size Exclusion Chromatography.


Acknowledgment is made to the donors of the American Chemical Society Petroleum Research Fund for support of this research (Grant no. 59 434-ND6). T.J.W. acknowledges partial funding from the Maryland Industrial Partnerships program.

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Correspondence to T. J. Woehl or M. A. Anisimov.

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Nikfarjam, S., Ghorbani, M., Adhikari, S. et al. Irreversible Nature of Mesoscopic Aggregates in Lysozyme Solutions. Colloid J 81, 546–554 (2019).

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