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Mixture of Macromolecular Crowding Agents Has a Non-additive Effect on the Stability of Proteins

  • Sumra Shahid
  • Faizan Ahmad
  • Md. Imtaiyaz Hassan
  • Asimul IslamEmail author
Article
  • 15 Downloads

Abstract

The folding and unfolding of proteins inside a cell take place in the presence of macromolecules of various shapes and sizes. Such crowded conditions can significantly affect folding, stability, and biophysical properties of proteins. Thus, to logically mimic the intracellular environment, the thermodynamic stability of two different proteins (lysozyme and α-lactalbumin) was investigated in the presence of mixtures of three crowding agents (ficoll 70, dextran 70, and dextran 40) at different pH values. These crowders possess different shapes and sizes. It was observed that the stabilizing effect of mixtures of crowders is more than the sum effects of the individual crowder, i.e., the stabilizing effect is non-additive in nature. Moreover, dextran 40 (in the mixture) has been found to exhibit the greatest stabilization when compared with other crowders in the mixture. In other words, the small size of the crowder has been observed to be a dominant factor in stabilization of the proteins.

Graphical Abstract

Keywords

Mixed macromolecular crowding Protein stability Crowder size Crowder shape Exclusion volume 

Abbreviations

GdmCl

Guanidinium chloride

UV

Ultra-violet

Tm

Midpoint of thermal denaturation

ΔHm

Enthalpy change at Tm

ΔCp

Constant-pressure heat capacity change

GD°

Gibbs free energy change at 25 °C

F70

Ficoll 70

D70

Dextran 70

D40

Dextran 40

Notes

Funding Information

This work was supported by grant from the Science & Engineering Research Board (SERB), India (SR/FT/LS-48/2010), FIST Program (SR/FST/LSI-541/2012), and Council of Scientific and Industrial Research (CSIR), India (37(1604)/13/EMR-II). SS is thankful to Maulana Azad National Fellowship, University Grants Commission (Government of India), for providing fellowship. FA is grateful to Indian National Science Academy for the award of Senior Scientist Position.

Supplementary material

12010_2019_2972_MOESM1_ESM.docx (28 kb)
ESM 1 (DOCX 28 kb)

References

  1. 1.
    Fulton, A. (1982). How crowded is the cytoplasm ? Cell, 30(2), 345–347.CrossRefGoogle Scholar
  2. 2.
    Minton, A. P. (1983). The effect of volume occupancy upon the thermodynamic activity of proteins: some biochemical consequences. Molecular and Cellular Biochemistry, 55(2), 119–140.CrossRefGoogle Scholar
  3. 3.
    Zimmerman, S. B., & Trach, S. O. (1991). Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. Journal of Molecular Biology, 222(3), 599–620.CrossRefGoogle Scholar
  4. 4.
    Medalia, O., Weber, I., Frangakis, A. S., Nicastro, D., Gerisch, G., & Baumeister, W. (2002). Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography. Science, 298(5596), 1209–1213.CrossRefGoogle Scholar
  5. 5.
    Ellis, R. J., & Minton, A. P. (2003). Cell biology: join the crowd. Nature, 425(6953), 27–28.CrossRefGoogle Scholar
  6. 6.
    Rivas, G., Ferrone, F., & Herzfeld, J. (2004). Life in a crowded world. EMBO Reports, 5(1), 23–27.CrossRefGoogle Scholar
  7. 7.
    Minton, A. P. (1981). Excluded volume as a determinant of macromolecular structure and reactivity. Biopolymers, 20(10), 2093–2120.CrossRefGoogle Scholar
  8. 8.
    Ellis, R. J. (2001). Macromolecular crowding: an important but neglected aspect of the intracellular environment. Current Opinion in Structural Biology, 11(1), 114–119.CrossRefGoogle Scholar
  9. 9.
    Zimmerman, S. B., & Minton, A. P. (1993). Macromolecular crowding: biochemical, biophysical, and physiological consequences. Annual Review of Biophysics and Biomolecular Structure, 22(1), 27–65.CrossRefGoogle Scholar
  10. 10.
    Ellis, R. J. (2001). Macromolecular crowding: obvious but underappreciated. Trends in Biochemical Sciences, 26(10), 597–604.CrossRefGoogle Scholar
  11. 11.
    Goodsell, D. S. (1991). Inside a living cell. Trends in Biochemical Sciences, 16(6), 203–206.CrossRefGoogle Scholar
  12. 12.
    Chebotareva, N. A., Kurganov, B. I., & Livanova, N. B. (2004). Biochemical effects of molecular crowding. Biochemistry (Moscow), 69(11), 1239–1251.CrossRefGoogle Scholar
  13. 13.
    Christiansen, A., Wang, Q., Cheung, M. S., & Wittung-Stafshede, P. (2013). Effects of macromolecular crowding agents on protein folding in vitro and in silico. Biophysical Reviews, 5(2), 137–145.CrossRefGoogle Scholar
  14. 14.
    Kuznetsova, I., Turoverov, K., & Uversky, V. (2014). What macromolecular crowding can do to a protein. International Journal of Molecular Sciences, 15(12), 23090–23140.CrossRefGoogle Scholar
  15. 15.
    Kuznetsova, I., Zaslavsky, B., Breydo, L., Turoverov, K., & Uversky, V. (2015). Beyond the excluded volume effects: mechanistic complexity of the crowded milieu. Molecules, 20(1), 1377–1409.CrossRefGoogle Scholar
  16. 16.
    Minton, A. P. (2001). The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media. The Journal of Biological Chemistry, 276(14), 10577–10580.CrossRefGoogle Scholar
  17. 17.
    Samiotakis, A., Wittung-Stafshede, P., & Cheung, M. S. (2009). Folding, stability and shape of proteins in crowded environments: experimental and computational approaches. International Journal of Molecular Sciences, 10(2), 572–588.CrossRefGoogle Scholar
  18. 18.
    Zhou, H.-X., Rivas, G., & Minton, A. P. (2008). Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Annual Review of Biophysics, 37(1), 375–397.CrossRefGoogle Scholar
  19. 19.
    Du, F., Zhou, Z., Mo, Z. Y., Shi, J. Z., Chen, J., & Liang, Y. (2006). Mixed macromolecular crowding accelerates the refolding of rabbit muscle creatine kinase: implications for protein folding in physiological environments. Journal of Molecular Biology, 364(3), 469–482.CrossRefGoogle Scholar
  20. 20.
    Fan, J., ZHOU, Z., Chen, J., YANG, Z.-Z. and Liang, Y. (2012) The contrasting effect of macromolecular crowding on protein misfolding. FEBS JOURNAL, pp. 408–408. WILEY-BLACKWELL 111 RIVER ST, HOBOKEN 07030–5774, NJ USA.Google Scholar
  21. 21.
    Zhou, B.-R., Liang, Y., Du, F., Zhou, Z., & Chen, J. (2004). Mixed macromolecular crowding accelerates the oxidative refolding of reduced, denatured lysozyme: implications for protein folding in intracellular environments. Journal of Biological Chemistry, 279(53), 55109–55116.CrossRefGoogle Scholar
  22. 22.
    Zhou, B. R., Zhou, Z., Hu, Q. L., Chen, J., & Liang, Y. (2008). Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme. Biochimica et Biophysica Acta, 1784(3), 472–480.CrossRefGoogle Scholar
  23. 23.
    Batra, J., Xu, K., & Zhou, H. X. (2009). Nonadditive effects of mixed crowding on protein stability. Proteins, 77(1), 133–138.CrossRefGoogle Scholar
  24. 24.
    Zhou, H. X. (2008). Effect of mixed macromolecular crowding agents on protein folding. Proteins, 72(4), 1109–1113.CrossRefGoogle Scholar
  25. 25.
    Privalov, P. L., & Khechinashvili, N. N. (1974). A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. Journal of Molecular Biology, 86(3), 665–684.CrossRefGoogle Scholar
  26. 26.
    Beg, I., Minton, A. P., Hassan, M. I., Islam, A., & Ahmad, F. (2015). Thermal stabilization of proteins by mono- and oligosaccharides: measurement and analysis in the context of an excluded volume model. Biochemistry, 54(23), 3594–3603.CrossRefGoogle Scholar
  27. 27.
    Beg, I., Minton, A. P., Islam, A., Hassan, M. I., & Ahmad, F. (2017). The pH dependence of saccharides’ influence on thermal denaturation of two model proteins supports an excluded volume model for stabilization generalized to allow for intramolecular electrostatic interactions. The Journal of Biological Chemistry, 292(2), 505–511.CrossRefGoogle Scholar
  28. 28.
    Beg, I., Minton, A. P., Islam, A., Hassan, M. I., & Ahmad, F. (2018). Comparison of the thermal stabilization of proteins by oligosaccharides and monosaccharide mixtures: measurement and analysis in the context of excluded volume theory. Biophysical Chemistry, 237, 31–37.CrossRefGoogle Scholar
  29. 29.
    Khan, S., Bano, Z., Singh, L. R., Hassan, M. I., Islam, A., & Ahmad, F. (2013). Testing the ability of non-methylamine osmolytes present in kidney cells to counteract the deleterious effects of urea on structure, stability and function of proteins. PLoS One, 8(9), e72533.CrossRefGoogle Scholar
  30. 30.
    Shahid, S., Ahmad, F., Hassan, M. I., & Islam, A. (2015). Relationship between protein stability and functional activity in the presence of macromolecular crowding agents alone and in mixture: an insight into stability-activity trade-off. Archives of Biochemistry and Biophysics, 584, 42–50.CrossRefGoogle Scholar
  31. 31.
    Singh, R., Haque, I., & Ahmad, F. (2005). Counteracting osmolyte trimethylamine N-oxide destabilizes proteins at pH below its pKa: measurements of thermodynamic parameters of proteins in the presence and absence of trimethylamine N-oxide. Journal of Biological Chemistry, 280(12), 11035–11042.CrossRefGoogle Scholar
  32. 32.
    Arakawa, T., & Timasheff, S. N. (1982). Stabilization of protein structure by sugars. Biochemistry, 21(25), 6536–6544.CrossRefGoogle Scholar
  33. 33.
    Mittal, S., & Singh, L. R. (2013). Denatured state structural property determines protein stabilization by macromolecular crowding: a thermodynamic and structural approach. PLoS One, 8(11), e78936.CrossRefGoogle Scholar
  34. 34.
    Sasahara, K., McPhie, P., & Minton, A. P. (2003). Effect of dextran on protein stability and conformation attributed to macromolecular crowding. Journal of Molecular Biology, 326(4), 1227–1237.CrossRefGoogle Scholar
  35. 35.
    Sharma, G. S., Mittal, S., & Singh, L. R. (2015). Effect of dextran 70 on the thermodynamic and structural properties of proteins. International Journal of Biological Macromolecules, 79, 86–94.CrossRefGoogle Scholar
  36. 36.
    Dumitriu, S. (2004). Polysaccharides: Structural diversity and functional versatility, second edition. CRC Press.Google Scholar
  37. 37.
    Luby-Phelps, K., Castle, P. E., Taylor, D. L., & Lanni, F. (1987). Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. Proceedings of the National Academy of Sciences of the United States of America, 84(14), 4910–4913.CrossRefGoogle Scholar
  38. 38.
    Venturoli, D. and Rippe, B. (2005) Ficoll and dextran vs. globular proteins as probes for testing glomerular permselectivity: effects of molecular size, shape, charge, and deformability. ed.Google Scholar
  39. 39.
    Hamaguchi, K., & Kurono, A. (1963). Structure of muramidase (lysozyme) I. The effect of guanidine hydrochloride on muramidase. The Journal of Biochemistry, 54, 111–122.Google Scholar
  40. 40.
    Sugai, S., Yashiro, H., & Nitta, K. (1973). Equilibrium and kinetics of the unfolding of α-lactalbumin by guanidine hydrochloride. Biochimica et Biophysica Acta (BBA) - Protein Structure, 328(1), 35–41.CrossRefGoogle Scholar
  41. 41.
    Nozaki, Y. (1972). The preparation of guanidine hydrochloride. Methods in Enzymology, 26, 43–50.CrossRefGoogle Scholar
  42. 42.
    A guide to multi-detector gel permeation chromatography 2012. Available from: www.agilent.com/chem.
  43. 43.
    Fissell, W. H., Hofmann, C. L., Smith, R., & Chen, M. H. (2010). Size and conformation of Ficoll as determined by size-exclusion chromatography followed by multiangle light scattering. American Journal of Physiology - Renal Physiology, 298(1), F205–F208.CrossRefGoogle Scholar
  44. 44.
    Sinha, A., Yadav, S., Ahmad, R., & Ahmad, F. (2000). A possible origin of differences between calorimetric and equilibrium estimates of stability parameters of proteins. Biochemical Journal, 345(3), 711–717.CrossRefGoogle Scholar
  45. 45.
    Yadav, S., & Ahmad, F. (2000). A new method for the determination of stability parameters of proteins from their heat-induced denaturation curves. Analytical Biochemistry, 283(2), 207–213.CrossRefGoogle Scholar
  46. 46.
    Becktel, W. J., & Schellman, J. A. (1987). Protein stability curves. Biopolymers, 26(11), 1859–1877.CrossRefGoogle Scholar
  47. 47.
    Hiraoka, Y., & Sugai, S. (1984). Thermodynamics of thermal unfolding of bovine apo-α-lactalbumin. International Journal of Peptide and Protein Research, 23(5), 535–542.CrossRefGoogle Scholar
  48. 48.
    Privalov, P. L. (1979). Stability of proteins: small globular proteins. Advances in Protein Chemistry, 33, 167–241.CrossRefGoogle Scholar
  49. 49.
    Homchaudhuri, L., Sarma, N., & Swaminathan, R. (2006). Effect of crowding by dextrans and Ficolls on the rate of alkaline phosphatase-catalyzed hydrolysis: a size-dependent investigation. Biopolymers, 83(5), 477–486.CrossRefGoogle Scholar
  50. 50.
    Shahid, S., Hassan, M. I., Islam, A., & Ahmad, F. (2017). Size-dependent studies of macromolecular crowding on the thermodynamic stability, structure and functional activity of proteins: in vitro and in silico approaches. Biochimica et Biophysica Acta (BBA) - General Subjects, 1861(2), 178–197.CrossRefGoogle Scholar
  51. 51.
    Alfano, C., Sanfelice, D., Martin, S. R., Pastore, A., & Temussi, P. A. (2017). An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states. Nature Communications, 8, 15428.CrossRefGoogle Scholar
  52. 52.
    Ghahghaei, A., & Mohammadian, S. (2014). The effect of Arg on the structure perturbation and chaperone activity of α-crystallin in the presence of the crowding agent, dextran. Applied Biochemistry and Biotechnology, 174(2), 739–750.CrossRefGoogle Scholar
  53. 53.
    Kumar, K., Bhargava, P., & Roy, U. (2011). In vitro refolding of Triosephosphate isomerase from L. donovani. Applied Biochemistry and Biotechnology, 164(7), 1207–1214.CrossRefGoogle Scholar
  54. 54.
    Fan, Y.-Q., Liu, H.-J., Li, C., Luan, Y.-S., Yang, J.-M., & Wang, Y.-L. (2013). Inactivation of recombinant human brain-type creatine kinase during denaturation by guanidine hydrochloride in a macromolecular crowding system. Applied Biochemistry and Biotechnology, 169(1), 268–280.CrossRefGoogle Scholar
  55. 55.
    Kumar, R., Sharma, D., Garg, M., Kumar, V., & Agarwal, M. C. (2018). Macromolecular crowding-induced molten globule states of the alkali pH-denatured proteins. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1866(11), 1102–1114.CrossRefGoogle Scholar
  56. 56.
    Benton, L. A., Smith, A. E., Young, G. B., & Pielak, G. J. (2012). Unexpected effects of macromolecular crowding on protein stability. Biochemistry, 51(49), 9773–9775.CrossRefGoogle Scholar
  57. 57.
    Charlton, L. M., Barnes, C. O., Li, C., Orans, J., Young, G. B., & Pielak, G. J. (2008). Residue-level interrogation of macromolecular crowding effects on protein stability. Journal of the American Chemical Society, 130(21), 6826–6830.CrossRefGoogle Scholar
  58. 58.
    Miklos, A. C., Li, C., Sharaf, N. G., & Pielak, G. J. (2010). Volume exclusion and soft interaction effects on protein stability under crowded conditions. Biochemistry, 49(33), 6984–6991.CrossRefGoogle Scholar
  59. 59.
    Miklos, A. C., Sarkar, M., Wang, Y., & Pielak, G. J. (2011). Protein crowding tunes protein stability. Journal of the American Chemical Society, 133(18), 7116–7120.CrossRefGoogle Scholar
  60. 60.
    Sarkar, M., Li, C., & Pielak, G. J. (2013). Soft interactions and crowding. Biophysical Reviews, 5(2), 187–194.CrossRefGoogle Scholar
  61. 61.
    Sarkar, M., Lu, J., & Pielak, G. J. (2014). Protein crowder charge and protein stability. Biochemistry, 53(10), 1601–1606.CrossRefGoogle Scholar
  62. 62.
    Sarkar, M., Smith, A. E., & Pielak, G. J. (2013). Impact of reconstituted cytosol on protein stability. Proceedings of the National Academy of Sciences, 110(48), 19342–19347.CrossRefGoogle Scholar
  63. 63.
    Schlesinger, A. P., Wang, Y., Tadeo, X., Millet, O., & Pielak, G. J. (2011). Macromolecular crowding fails to fold a globular protein in cells. Journal of the American Chemical Society, 133(21), 8082–8085.CrossRefGoogle Scholar
  64. 64.
    Wang, Y., Sarkar, M., Smith, A. E., Krois, A. S., & Pielak, G. J. (2012). Macromolecular crowding and protein stability. Journal of the American Chemical Society, 134(40), 16614–16618.CrossRefGoogle Scholar
  65. 65.
    Ignatova, Z., & Gierasch, L. M. (2004). Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling. Proceedings of the National Academy of Sciences of the United States of America, 101(2), 523–528.CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia IslamiaNew DelhiIndia

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