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J. Shi, H.A. Koteiche, P.L. Stewart, H.S. Mchaourab; Structure of an Activated Oligomeric Assembly of Hsp16.5 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2000.
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© ARVO (1962-2015); The Authors (2016-present)
Small heat shock proteins (sHSP) transition to activated states in response to elevated temperature and phosphorylation. However, the structural basis of the activation remains unclear. Studies of mammalian sHSP are hampered by their intrinsically dynamic oligomeric structure and their polydispersity. To overcome these difficulties, we have used cryo electron microscopy, EPR spectroscopy and binding assays to explore the plasticity of the oligomeric structure of the ordered Hsp16.5.
We constructed three mutants of Hsp16.5. Hsp16.5del is a truncation mutant that removes the N–terminal segment, Hsp16.5ins has a peptide insertion grafted from human Hsp27, and R107G is equivalent to the disease causing mutation in αB–crystallin, R120G. Cryo electron micrographs were collected of all three samples. 3D reconstructions were generated using single particle reconstruction methods. A series of destabilized T4 lysozyme (T4L) mutants labeled with bimane were used for binding analysis. Substrate concentrations were set to constant values, and the Hsp16.5 mutants concentrations varied. The mixtures are incubated at 37oC for 3 hr to reach equilibrium and the fluorescence emission measured on a PTI fluorometer.
Light scattering indicates that the Hsp16.5ins oligomer is a 48–mer and a 3D reconstruction shows that it has octahedral symmetry. The Hsp16.5del reconstruction shows that the number of subunits (24–mer) and symmetry is the same as WT. EPR analysis confirms that interactions between monomers related by 2–fold and 4–fold symmetry are preserved in the Hsp16.5ins oligomer, while the 3–fold symmetry interactions are severely distorted compared to WT. Preliminary docking of a dimer of α–crystallin domains from the Hsp16.5 crystal structure into the 3D reconstruction of Hsp16.5ins indicates that the N–terminal segments extend into the interior of the oligomer. A more detailed analysis of the new oligomeric assembly of Hsp16.5ins will be revealed by combining a sub–nanometer cryo–EM reconstruction with site–specific EPR information. The Hsp16.5del shows significant reduction in T4L binding while Hsp16.5ins displays higher affinity to T4L mutants relative to the WT.
The functional property of Hsp16.5ins suggests that it is an activated form of Hsp16.5. The increased size of the oligomer is consistent with the expansion upon substrate binding observed in other sHSP. Sub–nanometer resolution of this structure will illuminate important aspects of the interaction of sHSP with their substrates. The functional analysis of Hsp16.5del suggests an important role for the N–terminal segment in binding and recognition.
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