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J Dong, HA Koteiche, HS Mchaourab; Sequence Determinants of Quaternary Structure Flexibility in Hsp27 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3566.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:The N-terminal segments of small heat shock proteins (sHSPs) play an important role in the dynamics of their oligomeric structure. This quaternary structure flexibility, reflected in the phenomenon of subunit exchange, is essential for the function of these proteins. In this study, we explored the determinants of flexibility in the N-terminal domain of heat shock protein 27(Hsp27) and for reference determined the fold of its α-crystallin domain. Methods:The fold of the α-crystallin domain was determined using distance restraints obtained from pairs of spin labels. The sites of labeling were selected based on the structure of this domain in αA-crystallin. A D3 mutant (S15D, S78D, S82D) mimicking the effect of phosphorylation of S15, S78, S82 residues of Hsp27 was constructed. Single cysteine mutants were introduced at the same sites in the D3 and WT backgrounds. In addition, size exclusion chromatography (SEC) was used to monitor the oligomer equilibrium of WT Hsp27 and the various mutants. Results:The α-crystallin domain in Hsp 27 and its D3 variant have a similar structure to that of αA-crsytallin. However, in the region of the N-terminal domain just preceeding the α-crystallin domain, Electron Paramagnetic Resonance (EPR) spectroscopy reveals considerable conformational heterogeneity in WT Hsp 27. The same spectral features are not observed deep in the N-terminal domain or in the α-crystallin domain. SEC analysis suggests that the observed heterogeneity is a consequence of an equilibrium between two oligomeric species. The introduction of the D3 mutations, which shift the equilibrium towards a tetrameric form, affects the EPR spectral features in a consistent fashion. The mutagenic analysis also reveals single sites in the N-terminal domain that can reverse the effects of the D3 mutations. Conclusion:In the context of the two-domain paradigm of sHSP structure, the data defines two regions in the N-terminal domain of Hsp27 involved in the flexibility of the oligomeric structure. In the first, substitution of Asp or Glu significantly shifts the oligomeric equilibrium without changes in the local structure. In the second, the dissociation of the native oligomer is accompanied by a local conformational transition.
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