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J. A. King, K. L. Drahos; Mutations in the Buried Core of Human gD-Crystallin Sharply Destabilize the Native State. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5024.
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© ARVO (1962-2015); The Authors (2016-present)
Though cataracts represent aggregated states of damaged and misfolded forms of the lens crystallins, the actual conformational perturbations are poorly understood. Mutant crystallins that cause cataract can provide some insight into these processes. A set of amino acid substitutions in the buried core of the crystallin Greek key domains - L5S, V75D, & I90F - have been reported to be associated with congenital cataracts in mice. We have introduced these substitutions into the buried core of human gD-crystallin (HgD-Crys) to determine their effects on the conformation and stability of the human protein.
Mutant crystallins were generated in E. coli plasmids by site specific mutagenesis, and the resulting proteins expressed and purified from E. coli cultures. Protein conformation was determined using fluorescence spectroscopy by monitoring the emission from the four buried tryptophans of HgD-Crys. Stabilities of the mutant crystallins were assessed from equilibrium unfolding and refolding reactions at 37 °C using guanidine hydrochloride as the denaturing agent, in neutral buffer.
Human gD-Crys unfolds and refolds through a partially folded intermediate with the N-terminal domain disordered and the C-terminal domain native-like. The mutations L5S and V75D are found in the N-terminal domain and the mutation I90F is found in the C-terminal domain of the protein. The V75D and L5S proteins exhibited significant destabilization of their N-terminal domains, while their C-terminal domains retained native-like stability. The I90F protein showed a single two-state transition indicating that both domains were destabilized. This may represent the previously identified role of the domain interface in stabilizing the overall protein.
The three mutations which cause congenital cataracts in mice cause significant destabilization of human gD-Crys. L5S and V75D significantly destabilize the N-terminal domain of the protein, while I90F appears to destabilize both domains. Partially folded intermediates of all three proteins formed high molecular weight aggregates when diluted from denaturant to physiological buffer. These results indicate that damage to buried residues, whether by mutation, oxidation, or photo-oxidation, could seriously destabilize the native states of gD-crystallin, leading to aggregation.
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