Limits...
The congenital cataract-linked G61C mutation destabilizes γD-crystallin and promotes non-native aggregation.

Zhang W, Cai HC, Li FF, Xi YB, Ma X, Yan YB - PLoS ONE (2011)

Bottom Line: The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding.The aggregation-prone property of the mutant was not altered by the addition of reductive reagent.These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China.

ABSTRACT
γD-crystallin is one of the major structural proteins in human eye lens. The solubility and stability of γD-crystallin play a crucial role in maintaining the optical properties of the lens during the life span of an individual. Previous study has shown that the inherited mutation G61C results in autosomal dominant congenital cataract. In this research, we studied the effects of the G61C mutation on γD-crystallin structure, stability and aggregation via biophysical methods. CD, intrinsic and extrinsic fluorescence spectroscopy indicated that the G61C mutation did not affect the native structure of γD-crystallin. The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding. The mutation mainly affected the transition from the native state to the intermediate but not that from the intermediate to the unfolded or aggregated states. At high temperatures, both proteins were able to form aggregates, and the aggregation of the mutant was much more serious than the wild type protein at the same temperature. At body temperature and acidic conditions, the mutant was more prone to form amyloid-like fibrils. The aggregation-prone property of the mutant was not altered by the addition of reductive reagent. These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation.

Show MeSH

Related in: MedlinePlus

Effect of the G61C mutation on the unfolding of γD-crystallin induced by GdnHCl.Parameter A is the ratio of the intensity of the intrinsic fluorescence at 365 nm to that at 320 nm (I365/I320). The data were fitted to a three-state model, and the fitting results are shown by lines.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105094&req=5

pone-0020564-g006: Effect of the G61C mutation on the unfolding of γD-crystallin induced by GdnHCl.Parameter A is the ratio of the intensity of the intrinsic fluorescence at 365 nm to that at 320 nm (I365/I320). The data were fitted to a three-state model, and the fitting results are shown by lines.

Mentions: Equilibrium unfolding experiments were carried out to probe the effect of the mutation on the structural stability of γD-crystallin against chemical denaturants. Previous studies have shown that the unfolding of γD-crystallin undergoes a multi-state process with an intermediate populated under equilibrium unfolding conditions: native state (N)→intermediate (I)→unfolded state (U) [47], [48]. No off-pathway aggregation was observed when monitored by either turbidity at 400 nm or resonance Raleigh light scattering (data not shown). As presented in Figure 6, the unfolding of both the WT and mutated γD-crystallin was best-fitted by a three-state model when monitored by the intrinsic Trp fluorescence. The G61C mutation did not significantly affect the unfolding pathway of γD-crystallin. However, the midpoint of the N→I transition moved to a lower GdnHCl concentration, while the I→U transition was not significantly affected. Quantitatively, the midpoints of the N→I transition were 2.8 M and 2.0 M, and those of the I→U transition were 3.7 M and 3.6 M for the WT and G61C γD-crystallin, respectively. These results suggested that the G61C mutation significantly decreased the structural stability of the native state of γD-crystallin against chemical denaturants, but had little effect on the properties of the folding intermediate. This conclusion is consistent with the previous observation that the N→I transition of γD-crystallin denaturation induced by GdnHCl involves the unfolding of the N-terminal domain where G61 located [49].


The congenital cataract-linked G61C mutation destabilizes γD-crystallin and promotes non-native aggregation.

Zhang W, Cai HC, Li FF, Xi YB, Ma X, Yan YB - PLoS ONE (2011)

Effect of the G61C mutation on the unfolding of γD-crystallin induced by GdnHCl.Parameter A is the ratio of the intensity of the intrinsic fluorescence at 365 nm to that at 320 nm (I365/I320). The data were fitted to a three-state model, and the fitting results are shown by lines.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105094&req=5

pone-0020564-g006: Effect of the G61C mutation on the unfolding of γD-crystallin induced by GdnHCl.Parameter A is the ratio of the intensity of the intrinsic fluorescence at 365 nm to that at 320 nm (I365/I320). The data were fitted to a three-state model, and the fitting results are shown by lines.
Mentions: Equilibrium unfolding experiments were carried out to probe the effect of the mutation on the structural stability of γD-crystallin against chemical denaturants. Previous studies have shown that the unfolding of γD-crystallin undergoes a multi-state process with an intermediate populated under equilibrium unfolding conditions: native state (N)→intermediate (I)→unfolded state (U) [47], [48]. No off-pathway aggregation was observed when monitored by either turbidity at 400 nm or resonance Raleigh light scattering (data not shown). As presented in Figure 6, the unfolding of both the WT and mutated γD-crystallin was best-fitted by a three-state model when monitored by the intrinsic Trp fluorescence. The G61C mutation did not significantly affect the unfolding pathway of γD-crystallin. However, the midpoint of the N→I transition moved to a lower GdnHCl concentration, while the I→U transition was not significantly affected. Quantitatively, the midpoints of the N→I transition were 2.8 M and 2.0 M, and those of the I→U transition were 3.7 M and 3.6 M for the WT and G61C γD-crystallin, respectively. These results suggested that the G61C mutation significantly decreased the structural stability of the native state of γD-crystallin against chemical denaturants, but had little effect on the properties of the folding intermediate. This conclusion is consistent with the previous observation that the N→I transition of γD-crystallin denaturation induced by GdnHCl involves the unfolding of the N-terminal domain where G61 located [49].

Bottom Line: The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding.The aggregation-prone property of the mutant was not altered by the addition of reductive reagent.These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China.

ABSTRACT
γD-crystallin is one of the major structural proteins in human eye lens. The solubility and stability of γD-crystallin play a crucial role in maintaining the optical properties of the lens during the life span of an individual. Previous study has shown that the inherited mutation G61C results in autosomal dominant congenital cataract. In this research, we studied the effects of the G61C mutation on γD-crystallin structure, stability and aggregation via biophysical methods. CD, intrinsic and extrinsic fluorescence spectroscopy indicated that the G61C mutation did not affect the native structure of γD-crystallin. The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding. The mutation mainly affected the transition from the native state to the intermediate but not that from the intermediate to the unfolded or aggregated states. At high temperatures, both proteins were able to form aggregates, and the aggregation of the mutant was much more serious than the wild type protein at the same temperature. At body temperature and acidic conditions, the mutant was more prone to form amyloid-like fibrils. The aggregation-prone property of the mutant was not altered by the addition of reductive reagent. These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation.

Show MeSH
Related in: MedlinePlus