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J. A. King, I. Mills-Henry, L. Acosta-Sampson; Recognition by AlphaB-Crystallin Chaperone of C-Terminal Binding Regions in Human GammaD-Crystallin Substrates. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5803.
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© ARVO (1962-2015); The Authors (2016-present)
To identify the sites in partially folded human γD-crystallin recognized by the αB-crystallin chaperone during suppression of aggregation.
Human αB-crystallin and D-crystallin were expressed in, and purified from bacteria. Human α-crystallin, one of the ubiquitous crystallins in vertebrate lenses, is a polydisperse complex of ~ 800 kD consisting of two subunits (~20 kD) αA- and αB-crystallin (αA- and αB-Crys). Its chaperone activity involves suppressing aggregation of substrates by binding aggregation-prone species. γD-crystallin (γD-Crys), is a member of the βγ-crystallin family, localized in the lens nucleus. Aggregates isolated from mature-onset cataracts contain damaged and misfolded forms of γD-Crys.
Human γD-Crys refolds through the population of a partially folded intermediate in vitro, which has its N-terminal domain unfolded and its C-terminal domain folded. At high protein concentrations aggregation of this species competes with productive refolding. Our previous work showed that the HγD-Crys substrate in γD-αB complexes resembles this populated folding intermediate, with its N-terminal domain unfolded and its C-terminal domain folded. Using single domain constructs of HγD-Crys, we have found that the partially folded C-terminal domain construct (γD-Ctd) aggregates upon refolding, while the N-terminal domain construct (γD-Ntd) does not aggregate under similar conditions. Using chaperone constructs lacking tryptophans, we determined the fluorescence properties of the bound C-td substrate, and found it corresponded to a partially folded chain. This complex was stable upon storage.
HαB-Crys can suppress the aggregation of partially folded species of the γD-Ctd and forms stable γD-Ctd-αB complexes. The results suggest that exposure of buried strands of the C-terminal Greek key are the determinants of γ-crystallin recognition and strong binding. The stability of the substrate/chaperone complex would result in saturation of the chaperone population in the lens nucleus, which may help explain the age dependence of mature-onset cataracts.
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