Purchase this article with an account.
Calvin Vetter, Kate Halverson, Ido Almog, Larry L David, Kirsten J Lampi; Effect of surface deamidations on structure and stability of γS crystallins. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5689.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Surface deamidations in γS crystallins have been identified in vivo to be as much as 10-fold more prevalent in the nuclear insoluble fraction than the soluble one, indicating they may play a role in decreasing crystallin stability. In this study we investigated the effect of deamidations at N14 in the N-terminal extension, and at N76 and N143 on the surface of the N-and C-terminal domains, respectively.
Wild-type (WT), N14D, N76D, N143D, and N14D/N76D/N143D (3X) γS crystallins were recombinantly expressed and purified. Protein stability at 0.5 uM was determined by measuring fluorescence in increasing guanidine hydrochloride (GuHCl) after reaching equilibrium. Protein surface accessibility was determined via hydrogen-deuterium exchange (HDX) after subjecting the protein to 2.5M GuHCl at 30°C for increasing times. Thermal stability at 24 uM was determined by measuring turbidity at 405nm during incubation at 70°C.
The concentration for the midpoint of unfolding for WT was 2.5 M GuHCl with no significant differences between the WT and mutants; however, at the lower protein concentrations of less than 0.5 uM, N14D and N143D unfolded at lower GuHCl. Also, at 2.5 M GuHCl, an intermediate species with an increase in +85 Da was detected by HDX in the 3X after 2 min and in both N14D and N76D after 4 min. During heat denaturation, WT and N14D were most stable with the other mutants having significantly greater turbidity. The differences seen between heat and chemical denaturation reflect differences in their unfolding mechanisms.
Deamidations in γS-crystallin at residues N14, N76, and N143 subtly disrupted the proteins’ stability during chemical unfolding with more significant disruption during thermal denaturation. These results suggest a mechanism for insolubilization of deamidated γS, but also suggest an additional modification such as isomerization may be required. Future HDX experiments will examine which regions of deamidated γS undergo more rapid surface exposure and detect levels of isomerization at these sites. These experiments will allow modeling of potential γS denaturation mechanisms during cataract formation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only