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Kalyan S. Ghosh, Ajay Pande, Geo Rajan, Jayanti Pande; Substrate Binding to Alpha Crystallin Chaperone Prevents Metal-ion Binding. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4744.
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Alpha crystallins are molecular chaperones, but also bind Cu2+ (and Zn2+) ions, which enhances their chaperone activity. Moreover, sequestration of Cu2+ prevents metal-ion mediated oxidations. In the lens, α-crystallins also bind substrate proteins such as γ-crystallins, and free α-crystallin is progressively depleted as the lens ages. It is not known how the depletion of free α-crystallin affects Cu2+ (and Zn2+) binding. Here we investigate (a) the spectroscopic changes accompanying Cu2+ binding to human αA-crystallin (HAA), and (b) the Cu2+ (and Zn2+) binding ability of HAA in the chaperone complex with human γD-crystallin (HGD).
The HAA-Cu2+ complex was made by reacting CuCl2 with HAA and purified by passing it through a Superose-6 size-exclusion chromatography (SEC) column. Absorption, circular dichroism (CD) and Raman spectroscopic studies were carried out. HAA-HGD complexes were made by incubating mixtures of HAA and HGD (2:1, w/w) at 50°C and 55°C. Cu2+ and Zn2+ ion concentrations were determined by using an extrinsic dye 4-(2-pyridylazo) resorcinol (PAR).
(a) The HAA-Cu2+ complex shows two absorption bands around 370 and 600 nm. The species with an absorption band ~370 nm is unstable and precipitates, but a stable complex containing ~0.5 mole of tightly bound Cu2+ per mole HAA monomer is obtained by SEC or extensive dialysis. The CD spectrum of the HAA-Cu2+ complex shows a positive band (~575 nm, d-d transition), and a negative band (~300 nm, charge-transfer). The dissociation constant (Kd) of the HAA-Cu2+ obtained from CD studies is 1.7x10-12 M. Raman spectral changes of the Cu2+ complex suggest that Cu2+ is liganded to His residues in HAA through the Nπ and Nτ atoms. (b) Binding of HGD, and of Cu2+ (and Zn2+) ions to HAA are inversely related: Higher the amount of HGD bound, lower the amount of bound metal ions.
1) Spectroscopic characterization of the stable HAA-Cu2+ complex shows detailed electronic and molecular structure 2) The chaperone complexes of α-crystallins show reduced Cu2+ (and Zn2+) binding ability.
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