Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 281, Issue 41, Pages 30782-30793Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M603882200
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- NIGMS NIH HHS [GM17980] Funding Source: Medline
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Human eye lens transparency requires life long stability and solubility of the crystallin proteins. Aged crystallins have high levels of covalent damage, including glutamine deamidation. Human gamma D-crystallin (H gamma D-Crys) is a two-domain beta-sheet protein of the lens nucleus. The two domains interact through interdomain side chain contacts, including Gln-54 and Gln-143, which are critical for stability and folding of the N-terminal domain of H gamma D-Crys. To test the effects of interface deamidation on stability and folding, single and double glutamine to glutamate substitutions were constructed. Equilibrium unfolding/refolding experiments of the proteins were performed in guanidine hydrochloride at pH 7.0, 37 degrees C, or urea at pH 3.0, 20 degrees C. Compared with wild type, the deamidation mutants were destabilized at pH 7.0. The proteins populated a partially unfolded intermediate that likely had a structured C-terminal domain and unstructured N-terminal domain. However, at pH 3.0, equilibrium unfolding transitions of wild type and the deamidation mutants were indistinguishable. In contrast, the double alanine mutant Q54A/Q143A was destabilized at both pH 7.0 and 3.0. Thermal stabilities of the deamidation mutants were also reduced at pH 7.0. Similarly, the deamidation mutants lowered the kinetic barrier to unfolding of the N-terminal domain. These data indicate that interface deamidation decreases the thermodynamic stability of H gamma D-Crys and lowers the kinetic barrier to unfolding due to introduction of a negative charge into the domain interface. Such effects may be significant for cataract formation by inducing protein aggregation or insolubility.
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