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J. Shi, P. L. Stewart, H. S. Mchaourab; Structure of Truncated Hsp16.5 in Complex With T4 Lysozyme. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2042.
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© ARVO (1962-2015); The Authors (2016-present)
To further develop the mechanistic understanding of small heat-shock protein chaperone activity, we determined the structure of a variant of Hsp16.5 complexed with the model substrate T4 Lysozyme using cryo electron microscopy.
For these studies we used Hsp16.5tr which is a variant that lacks the N-terminal region of 33 amino acids. Previous studies have shown that this mutant binds T4 lysozyme destabilized mutant with significant lower affinity than the WT. Complexes with T4L were prepared by incubation at 37oC for 3 hr to reach equilibrium and the binding was verified by analysis on size exclusion chromatography. Cryoelectron micrographs were collected on an FEI Tecnai 12 (120kV) cryoelectron microscope.
3D reconstructions were generated using single particle reconstruction methods for both uncomplexed Hsp16.5tr and Hsp16.5tr/T4L at ~10Å resolution. Approximately 20,000 particle images were included in each 3D reconstruction. The cryoEM reconstruction of Hsp16.5tr/T4L shows significant density in the interior of the octahedral assembly compared to that of uncomplexed Hsp16.5tr. At the current resolution, the internal density assigned to bound T4L does not seem to be in contact with the α-crystallin domain.
The data demonstrate that T4L binds on the inside of the Hsp16.5tr oligomer. The absence of detectable contacts with the outer shell suggests that these contacts cannot be extensive. Limited binding to the α-crystallin domain may serve to guide the unfolded state to the inner region of the oligomer. In the WT-Hsp16.5, interaction with the N-terminal region serves to stabilize the substrate and enhances the affinity.
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