Attempt to optimize some properties of fluorescent chimeras of human small heat shock protein HspB1 by modifying linker length and nature
- 71 Downloads
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 wordssmall heat shock proteins HspB1 fluorescent chimeras quaternary structure chaperone-like activity
enhanced yellow fluorescent protein
- sHsp (or HspB)
small heat shock proteins
Unable to display preview. Download preview PDF.
- 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
- 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.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
- 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.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.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