Cataract-causing mutation R233H affects the stabilities of βB1- and βA3/βB1-crystallins with different pH-dependence

• R233H stabilizes βB1 against guanidine hydrochloride-induced unfolding.
• R233H weakens the protection effect of βB1 on βA3 aggregation.
• The inter-subunit ion-pairing network of βB1 is intrinsically highly dynamic.
• The mutational effects of R233H on βB1 and βA3/βB1 have different pH-dependence.
• His protonation plays a regulatory role in crystallin stability and aggregation.

Disease-causing mutations can be stabilizing or destabilizing. Missense mutations of structural residues are generally destabilizing, while stabilizing mutations are usually linked to alterations in protein functions. Stabilizing mutations are rarely identified in mutations linked to congenital cataract, a disease caused by the opacification of the lens. In this research, we found that R233H mutation had little impact on βB1-crystallin structure, solubility and thermal stability under neutral solution pH conditions. The mutation increased βB1 stability against guanidine hydrochloride-induced denaturation, suggesting that Arg233 might be a functional residue. Further analysis indicated that the R233H mutation did not affect the formation of βA3/βB1 heteromer, but significantly reduced heteromer stability against heat- and guanidine hydrochloride-induced denaturation. The R233H mutation negatively affected the thermal stabilities and aggregatory propensities of βB1 and βA3/βB1 with different pH-dependence, implying that the protonation of His side chains during acidification played a regulatory role in crystallin stability and aggregation. Molecular dynamic simulations indicated that Arg233 is one of the residues forming an inter-subunit ion-pairing network with intrinsically dynamic nature. Based on these observations, we proposed that the highly dynamic ion-pairing network contributed to the tradeoff among βB1 solubility, stability, aggregatory propensity and function of protecting βA3.

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