Glutamine deamidation destabilizes human γD-crystallin and lowers the kinetic barrier to unfolding
Glutamine deamidation destabilizes human γD-crystallin and lowers the kinetic barrier to unfolding
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DOI：
10.1074/jbc.m603882200
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发表时间：
2006-10-13
影响因子：
4.8
通讯作者：
King, Jonathan
中科院分区：
文献类型：
--
作者：
Flaugh, Shannon L.;Mills, Ishara A.;King, Jonathan
人眼透镜的透明度需要晶状体蛋白的终身稳定性和溶解性。老化的晶状体蛋白具有高水平的共价损伤，包括谷氨酰胺脱酰胺。人γ D-晶状体蛋白（H gamma D-Crys）是透镜核的双结构域β折叠蛋白。这两个结构域通过结构域间侧链接触相互作用，包括Gln-54和Gln-143，这对H γ D-Crys的N-末端结构域的稳定性和折叠至关重要。为了测试界面脱酰胺对稳定性和折叠的影响，构建了单谷氨酰胺和双谷氨酰胺至谷氨酸盐的取代。蛋白质的平衡解折叠/重折叠实验在pH 7.0，37 ℃的盐酸胍或pH 3.0，20 ℃的尿素中进行。与野生型相比，脱酰胺突变体在pH 7.0下不稳定。这些蛋白质占据了一个部分未折叠的中间体，该中间体可能具有结构化的C-末端结构域和非结构化的N-末端结构域。然而，在pH 3.0时，野生型和脱酰胺突变体的平衡解折叠转变是不可区分的。相反，双丙氨酸突变体Q54 A/Q143 A在pH 7.0和3.0下都不稳定。脱酰胺突变体的热稳定性也降低在pH 7.0。类似地，脱酰胺突变体降低了N-末端结构域展开的动力学屏障。这些数据表明，界面脱酰胺降低了H γ D-Crys的热力学稳定性，并降低了由于引入负电荷到域界面中而展开的动力学势垒。这种作用可能通过诱导蛋白质聚集或不溶性而对白内障形成具有重要意义。
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.