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E. T. McDonald; Structure of the Exchanging Unit of Hsp 27. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2043. doi: https://doi.org/.
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α-crystallins and Hsp27 form polydisperse oligomers that undergo subunit exchange. The process involves the reversible dissociation of groups of subunit. We are using spectroscopic tools to determine structural features of the dissociated multimer.
For this purpose, we introduced cysteines at a number of sites across the Hsp27 subunit either in the WT background or in a background where three serine residues were substituted by aspartates to mimic the fully phosphorylated form of Hsp27. The cysteine mutants were expressed and purified and subsequently reacted with a sulfhydryl specific spin label. Spin labeled Hsp27 mutants were analyzed by electron paramagnetic resonance (EPR) spectroscopy and gel filtration chromatography.
Hsp27 dissociation equilibrium is modulated by protein concentration, temperature, pH as well as phosphorylation. The latter favors the dissociated states to the extent that triply phosphorylated Hsp27 is predominantly a multimer over a wide range of concentration. Structural transitions upon phosphorylation were detected by comparing the EPR spectra of spin labeled mutants in the WT background and the phsophorylation mimic. Our data reveals a substantial conformational change in the N-terminal domain and in a flexible peptide linker at the junction between the N- and C-terminal domains. In the latter, EPR spectra characteristics of dynamic disorder suggest a highly unfolded backbone. Residues in the N-terminal domain report a transition to a more solvent-exposed environment although the backbone remains structured. The folding pattern of the α-crystallin domain remains intact in the phosphorylation mimic as revealed by the preservation of spin label proximities between neighboring ß-strands.
Previous work from our laboratory correlated the phosphorylation-induced dissociation of Hsp27 with increased level of substrate binding by Hsp27. Given the lack of substantial rearrangements in the α-crystallin domain, our results suggest that the structural basis of enhanced substrate binding is the exposure of the N-terminal domain.
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