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Jayanti Pande, Natalya Mokhor, Ajay Pande; Modeling the effect of extensive deamidation of gammaS-crystallin on aging and cataract formation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5594.
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© ARVO (1962-2015); The Authors (2016-present)
Deamidation of proteins upon aging appears to be a universal phenomenon. The effect of deamidation on aging and cataract formation has been extensively studied for some members of the lens crystallin family. Here we examine in vitro, how the deamidation of specific Asn (N76 and 143) and Gln (Q92 and 120) residues of human gammaS-crystallin known to be associated with aging and cataract formation, affect the structure and solution properties of the protein.
Human gammaS-crystallin and two mutants were expressed in E.Coli: Mutant 2N corresponds to the replacement of Asn 76 and 143 to Asp, and mutant 2N2Q contains two additional mutations, Gln 92 and 120 to Glu. Thus, 2N2Q represents an extensively deamidated product. Isoelectric Focusing and SDS-PAGE were used to characterize the mutants. Spectroscopic measurements (Circular Dichroism and Tryptophan Fluorescence emission) were carried out to determine the secondary and tertiary structures of the mutants, and Guanidinium Hydrochloride-mediated unfolding was used to determine their relative stabilities. Light scattering measurements were carried out to determine the strength and nature of the protein-protein interactions in the deamidated crystallins relative to the wild-type.
Both deamidated mutants show normal solubility. The pI values of the 2N and 2N2Q mutants were lowered by about 0.5 and 0.8 pH units respectively relative to that of wild-type gammaS-crystallin. All three proteins showed a predominantly single band on the IEF gel only in the presence of DTT. The structures and stabilities of both deamidated derivatives are comparable to that of the wild-type protein. Light-scattering measurements indicate that the repulsive protein-protein interactions observed in the wild-type are compromised in the mutants. This may lead to a propensity for aggregation upon deamidation.
Extensive deamidation does not significantly affect the secondary and tertiary structure of human gammaS-crystallin, but may alter protein-protein interactions. PEG-induced phase transition studies are currently being conducted to substantiate these findings.
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