Advertisement

Biochemistry (Moscow)

, Volume 80, Issue 1, pp 67–73 | Cite as

Attempt to optimize some properties of fluorescent chimeras of human small heat shock protein HspB1 by modifying linker length and nature

  • P. N. Datskevich
  • L. K. Muranova
  • N. B. GusevEmail author
Article

Abstract

Chimerical proteins consisting of enhanced yellow fluorescent protein (EYFP) connected by linkers of different length and nature to the N-terminal end of small heat shock protein HspB1 were obtained and characterized. To obtain fluorescent chimeras with properties similar to those of unmodified small heat shock protein, we used either 12-residue-long linkers of different nature (highly flexible Gly-Ser linker (L1), rigid α-helical linker (L2), or rigid Pro-Ala linker (L3)) or highly flexible Gly-Ser linker consisting of 12, 18, or 21 residues. The wild-type HspB1 formed large stable oligomers consisting of more than 20 subunits. Independent of the length or the nature of the linker, all the fluorescent chimeras formed small (5–9 subunits) oligomers tending to dissociate at low protein concentration. Chaperone-like activity of the wild-type HspB1 and its fluorescent chimeras were compared using lysozyme as a model protein substrate. Under the conditions used, all the fluorescent chimeras possessed higher chaperone-like activity than the wild-type HspB1. Chaperone-like activity of fluorescent chimeras with L1 and L3 linkers was less different from that of the wild-type HspB1 compare to the chaperone-like activity of chimeras with rigid L2 linker. Increase in the length of L1 linker from 12 up to 21 residues leads to decrease in the difference in the chaperone-like activity between the wild-type protein and its fluorescent chimeras. Since the N-terminal domain of small heat shock proteins participates in formation of large oligomers, any way of attachment of fluorescent protein to the N-terminal end of HspB1 leads to dramatic changes in its oligomeric structure. Long flexible linkers should be used to obtain fluorescent chimeras with chaperone-like properties similar to those of the wild-type HspB1.

Key words

small heat shock proteins HspB1 fluorescent chimeras quaternary structure chaperone-like activity 

Abbreviations

EYFP

enhanced yellow fluorescent protein

sHsp (or HspB)

small heat shock proteins

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Mymrikov, E. V., Seit-Nebi, A. S., and Gusev, N. B. (2011) Large potentials of small heat shock proteins, Physiol. Rev., 91, 1123–1159.PubMedCrossRefGoogle Scholar
  2. 2.
    Carra, S., Rusmini, P., Crippa, V., Giorgetti, E., Boncoraglio, A., Cristofani, R., Naujock, M., Meister, M., Minoia, M., Kampinga, H. H., and Poletti, A. (2013) Different anti-aggregation and pro-degradative functions of the members of the mammalian sHSP family in neurological disorders, Philos. Trans. R. Soc. Lond. B. Biol. Sci., 368.Google Scholar
  3. 3.
    Boelens, W. C., Croes, Y., and de Jong, W. W. (2001) Interaction between alphaB-crystallin and the human 20S proteasomal subunit C8/alpha7, Biochim. Biophys. Acta, 1544, 311–319.PubMedCrossRefGoogle Scholar
  4. 4.
    Paul, C., Simon, S., Gibert, B., Virot, S., Manero, F., and Arrigo, A. P. (2010) Dynamic processes that reflect antiapoptotic strategies set up by HspB1 (Hsp27), Exp. Cell Res., 316, 1535–1552.PubMedCrossRefGoogle Scholar
  5. 5.
    Crowe, J., Aubareda, A., McNamee, K., Przybycien, P. M., Lu, X., Williams, R. O., Bou-Gharios, G., Saklatvala, J., and Dean, J. L. (2013) Heat shock protein B1-deficient mice display impaired wound healing, PLoS ONE, 8, e77383.PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Wettstein, G., Bellaye, P. S., Micheau, O., and Bonniaud, P. (2012) Small heat shock proteins and the cytoskeleton: an essential interplay for cell integrity? Int. J. Biochem. Cell Biol., 44, 1680–1686.PubMedCrossRefGoogle Scholar
  7. 7.
    Seit-Nebi, A. S., Datskevich, P., and Gusev, N. B. (2013) Commentary on paper: “Small heat shock proteins and the cytoskeleton: an essential interplay for cell integrity?” (Wettstein et al.), Int. J. Biochem. Cell Biol., 45, 344–346.PubMedCrossRefGoogle Scholar
  8. 8.
    Datskevich, P. N., Nefedova, V. V., Sudnitsyna, M. V., and Gusev, N. B. (2012) Mutations of small heat shock proteins and human congenital diseases, Biochemistry (Moscow), 77, 1500–1514.CrossRefGoogle Scholar
  9. 9.
    Bagneris, C., Bateman, O. A., Naylor, C. E., Cronin, N., Boelens, W. C., Keep, N. H., and Slingsby, C. (2009) Crystal structures of alpha-crystallin domain dimers of alphaB-crystallin and Hsp20, J. Mol. Biol., 392, 1242–1252.PubMedCrossRefGoogle Scholar
  10. 10.
    Delbecq, S. P., and Klevit, R. E. (2013) One size does not fit all: the oligomeric states of alphaB crystallin, FEBS Lett., 587, 1073–1080.PubMedCrossRefGoogle Scholar
  11. 11.
    Lelj-Garolla, B., and Mauk, A. G. (2012) Roles of the N- and C-terminal sequences in Hsp27 self-association and chaperone activity, Protein Sci., 21, 122–133.PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Kappe, G., Franck, E., Verschuure, P., Boelens, W. C., Leunissen, J. A., and de Jong, W. W. (2003) The human genome encodes 10 alpha-crystallin-related small heat shock proteins: HspB1-10, Cell Stress Chaperones, 8, 53–61.PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Arrigo, A. P., and Gibert, B. (2012) HspB1 dynamic phospho-oligomeric structure dependent interactome as cancer therapeutic target, Curr. Mol. Med., 12, 1151–1163.PubMedCrossRefGoogle Scholar
  14. 14.
    Cox, D., Carver, J. A., and Ecroyd, H. (2014) Preventing alpha-synuclein aggregation: the role of the small heatshock molecular chaperone proteins, Biochim. Biophys. Acta, 1842, 1830–1843.PubMedCrossRefGoogle Scholar
  15. 15.
    Sun, X., Fontaine, J. M., Rest, J. S., Shelden, E. A., Welsh, M. J., and Benndorf, R. (2004) Interaction of human HSP22 (HSPB8) with other small heat shock proteins, J. Biol. Chem., 279, 2394–2402.PubMedCrossRefGoogle Scholar
  16. 16.
    Fontaine, J. M., Sun, X., Benndorf, R., and Welsh, M. J. (2005) Interactions of HSP22 (HSPB8) with HSP20, alphaB-crystallin, and HSPB3, Biochem. Biophys. Res. Commun., 337, 1006–1011.PubMedCrossRefGoogle Scholar
  17. 17.
    Fontaine, J. M., Sun, X., Hoppe, A. D., Simon, S., Vicart, P., Welsh, M. J., and Benndorf, R. (2006) Abnormal small heat shock protein interactions involving neuropathy-associated HSP22 (HSPB8) mutants, FASEB J., 20, 2168–2170.PubMedCrossRefGoogle Scholar
  18. 18.
    Borrelli, M. J., Bernock, L. J., Landry, J., Spitz, D. R., Weber, L. A., Hickey, E., Freeman, M. L., and Corry, P. M. (2002) Stress protection by a fluorescent Hsp27 chimera that is independent of nuclear translocation or multimeric dissociation, Cell Stress Chaperones, 7, 281–296.PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Evgrafov, O. V., Mersiyanova, I., Irobi, J., Van Den Bosch, L., Dierick, I., Leung, C. L., Schagina, O., Verpoorten, N., Van Impe, K., Fedotov, V., Dadali, E., et al. (2004) Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy, Nat. Genet., 36, 602–606.PubMedCrossRefGoogle Scholar
  20. 20.
    Delbecq, S. P., Jehle, S., and Klevit, R. (2012) Binding determinants of the small heat shock protein, alphaB-crystallin: recognition of the “IxI” motif, EMBO J., 31, 4587–4594.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Datskevich, P. N., Mymrikov, E. V., Sluchanko, N. N., Shemetov, A. A., Sudnitsyna, M. V., and Gusev, N. B. (2012) Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins, Protein Exp. Purif., 82, 45–54.CrossRefGoogle Scholar
  22. 22.
    Datskevich, P. N., Mymrikov, E. V., and Gusev, N. B. (2012) Utilization of fluorescent chimeras for investigation of heterooligomeric complexes formed by human small heat shock proteins, Biochimie, 94, 1794–1804.PubMedCrossRefGoogle Scholar
  23. 23.
    Datskevich, P. N., and Gusev, N. B. (2014) Structure and properties of chimeric small heat shock proteins containing yellow fluorescent protein attached to their C-terminal ends, Cell Stress Chaperones, 19, 507–518.PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Chen, X., Zaro, J. L., and Shen, W. C. (2013) Fusion protein linkers: property, design and functionality, Adv. Drug Deliv. Rev., 65, 1357–1369.PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Arai, R., Ueda, H., Kitayama, A., Kamiya, N., and Nagamune, T. (2001) Design of the linkers which effectively separate domains of a bifunctional fusion protein, Protein Eng., 14, 529–532.PubMedCrossRefGoogle Scholar
  26. 26.
    Arrigo, A. P. (2013) Human small heat shock proteins: protein interactomes of homo- and hetero-oligomeric complexes: an update, FEBS Lett., 587, 1959–1969.PubMedCrossRefGoogle Scholar
  27. 27.
    Shcherbo, D., Souslova, E. A., Goedhart, J., Chepurnykh, T. V., Gaintzeva, A., Shemiakina, I. I., Gadella, T. W., Lukyanov, S., and Chudakov, D. M. (2009) Practical and reliable FRET/FLIM pair of fluorescent proteins, BMC Biotechnol., 9, 24.PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Jehle, S., Vollmar, B. S., Bardiaux, B., Dove, K. K., Rajagopal, P., Gonen, T., Oschkinat, H., and Klevit, R. E. (2011) N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity, Proc. Natl. Acad. Sci. USA, 108, 6409–6414.PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Shashidharamurthy, R., Koteiche, H. A., Dong, J., and McHaourab, H. S. (2005) Mechanism of chaperone function in small heat shock proteins: dissociation of the HSP27 oligomer is required for recognition and binding of destabilized T4 lysozyme, J. Biol. Chem., 280, 5281–5289.PubMedCrossRefGoogle Scholar
  30. 30.
    Leder, L., Stark, W., Freuler, F., Marsh, M., Meyerhofer, M., Stettler, T., Mayr, L. M., Britanova, O. V., Strukova, L. A., Chudakov, D. M., and Souslova, E. A. (2010) The structure of Ca2+ sensor Case16 reveals the mechanism of reaction to low Ca2+ concentrations, Sensors, 10, 8143–8160.PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Ghosh, J. G., Estrada, M. R., and Clark, J. I. (2005) Interactive domains for chaperone activity in the small heat shock protein, human alphaB crystallin, Biochemistry, 44, 14854–14869.PubMedCrossRefGoogle Scholar
  32. 32.
    Jaya, N., Garcia, V., and Vierling, E. (2009) Substrate binding site flexibility of the small heat shock protein molecular chaperones, Proc. Natl. Acad. Sci. USA, 106, 15604–15609.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2015

Authors and Affiliations

  • P. N. Datskevich
    • 1
  • L. K. Muranova
    • 1
  • N. B. Gusev
    • 1
    Email author
  1. 1.Biological FacultyLomonosov Moscow State UniversityMoscowRussia

Personalised recommendations