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S.L. Flaugh, M.S. Kosinski–Collins, J.A. King; The Role of the Domain Interface of Human gammaD Crystallin in Folding, Stability and Fibril Formation . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3970.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Human gammaD crystallin (HgD–Crys) is a monomeric, two domain protein present in the lens nucleus and is involved in cataractogenesis. The interface between the two domains is thought to be important in the stability of the protein. Amino acid side chain interactions at the domain interface are characterized by a central hydrophobic cluster and peripheral interactions between Gln54 and Gln142 and Arg79 and Met146, which form a boundary between the hydrophobic cluster and the solvent. Glutamine residues have been found to be targets of deamidation in cataractous lenses. In this study, we address the contributions of the peripheral interactions to the folding, stability and in vitro fibril formation of HgD–Crys. Methods:Single and double alanine substitutions of Gln54, Gln142, Arg79, and Met146 were constructed, the mutant proteins expressed in E. coli and purified by Nickel affinity chromatography. Equilibrium unfolding/refolding in GuHCl was performed monitoring fluorescence emission. Thermal denaturation was monitored by circular dichroism spectroscopy at increasing temperatures. Results:All mutant proteins behaved as soluble subunits similar to wild type HgD–Crys during purification. The mutant proteins appeared native–like by circular dichroism, UV and fluorescence spectroscopy. All of the mutant proteins could be refolded after GuHCl denaturation. Equilibrium unfolding and refolding experiments revealed a single major transition for all mutant proteins with a midpoint of transition in the range of 2.63 to 2.70 M GuHCl. These midpoints were only slightly decreased compared to wild type which had a midpoint of 2.8 M GuHCl. All mutant proteins displayed an off–pathway aggregation reaction competing with productive refolding, similar to wild type. The range of [GuHCl] over which the transition occurred was increased for all mutant proteins. This difference is reflected in an altered slope of ΔG vs. [GuHCl] (m–value) for the transition region, suggestive of a decrease in the surface area of buried hydrophobics. The double mutation Q54A/Q142A had the most significant effect on the m–value, decreasing it from 4.9 for wild type to 2.27. Thermal denaturation experiments revealed a TM for wild type of 82 °C. All mutant proteins displayed decreased TM’s. Conclusions:The peripheral domain interface interactions are significant to the folding and stability of HgD–Crys. These sites may be important for shielding the internal hydrophobic cluster from solvent. Disruptions of the domain interface may generate the partially unfolded intermediates which are aggregation prone.
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