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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.

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Fitting of the intrinsic fluorescence spectra of the WT γD-crystallin recorded at 25°C (A), 75°C (B) and 83°C (C) by the discrete state model of Trp residues in proteins.The fitted spectra are the sum of the four spectral components: Class A & S centered at ∼318 nm is from the fluorophores in highly hydrophobic and rigid microenvironments, Class I at ∼330 nm reflect buried fluorophores inaccessible to solvent, Class II at ∼340 nm is assigned to fluorophores exposed to bound water, and Class III fluorophores at ∼350 nm are highly exposed to solvent [46]. The experimental data are shown as dotted lines. (D) Thermal dependence of the peak areas of the four fluorophores.
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pone-0020564-g005: Fitting of the intrinsic fluorescence spectra of the WT γD-crystallin recorded at 25°C (A), 75°C (B) and 83°C (C) by the discrete state model of Trp residues in proteins.The fitted spectra are the sum of the four spectral components: Class A & S centered at ∼318 nm is from the fluorophores in highly hydrophobic and rigid microenvironments, Class I at ∼330 nm reflect buried fluorophores inaccessible to solvent, Class II at ∼340 nm is assigned to fluorophores exposed to bound water, and Class III fluorophores at ∼350 nm are highly exposed to solvent [46]. The experimental data are shown as dotted lines. (D) Thermal dependence of the peak areas of the four fluorophores.

Mentions: γD-crystallin contains four Trp residues. When fitted by the discrete state model [42], [46], the intrinsic Trp fluorescence spectrum of the native γD-crystallin contains two major fluorophores, the Class A & S and Class I fluorophores, which centered at ∼318 nm and 330 nm, respectively (Figure 5). The peak area ratio was close to 1∶1 for the two fluorophores. The previous mutational analysis indicates that the fluorescence emission is significantly quenched for the W69-only and W157-only mutants, and the emission maximum is centered at 327 nm and 318 nm for the W43-only and W131-only mutants, respectively [47]. Thus it seems that in the fluorescence of native γD-crystallin, the Class A & S fluorophore was mainly contributed by W131, and the Class I fluorophore was mainly by W43. The existence of a minor content of Class II fluorophores centered at 340 nm may arise from the fast exchange of protein conformations in solutions. At 75°C where the intermediate state appeared during the thermal unfolding of the WT γD-crystallin, the contribution of the Class A & S fluorophore decreased, while that of the Class I fluorophore increased correspondingly. This may result from an alteration in the microenvironments of W131 in the intermediate state or an additional contribution by the Trp residues that are quenched in the native state. At temperatures above 75°C, the contents of both the Class A & S and Class I fluorophores decreased, while that of Class II increased continuously. This suggested that the microenvironments of all Trp residues were changed to the more solvent-accessible state upon heating, which is confirmed by the simultaneous increase in the hydrophobic exposure as monitored by ANS fluorescence (Figure 3C). Similar results were obtained for the mutated γD-crystallin except that the transitions occurred at a relatively lower temperature when compared with the WT γD-crystallin.


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)

Fitting of the intrinsic fluorescence spectra of the WT γD-crystallin recorded at 25°C (A), 75°C (B) and 83°C (C) by the discrete state model of Trp residues in proteins.The fitted spectra are the sum of the four spectral components: Class A & S centered at ∼318 nm is from the fluorophores in highly hydrophobic and rigid microenvironments, Class I at ∼330 nm reflect buried fluorophores inaccessible to solvent, Class II at ∼340 nm is assigned to fluorophores exposed to bound water, and Class III fluorophores at ∼350 nm are highly exposed to solvent [46]. The experimental data are shown as dotted lines. (D) Thermal dependence of the peak areas of the four fluorophores.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020564-g005: Fitting of the intrinsic fluorescence spectra of the WT γD-crystallin recorded at 25°C (A), 75°C (B) and 83°C (C) by the discrete state model of Trp residues in proteins.The fitted spectra are the sum of the four spectral components: Class A & S centered at ∼318 nm is from the fluorophores in highly hydrophobic and rigid microenvironments, Class I at ∼330 nm reflect buried fluorophores inaccessible to solvent, Class II at ∼340 nm is assigned to fluorophores exposed to bound water, and Class III fluorophores at ∼350 nm are highly exposed to solvent [46]. The experimental data are shown as dotted lines. (D) Thermal dependence of the peak areas of the four fluorophores.
Mentions: γD-crystallin contains four Trp residues. When fitted by the discrete state model [42], [46], the intrinsic Trp fluorescence spectrum of the native γD-crystallin contains two major fluorophores, the Class A & S and Class I fluorophores, which centered at ∼318 nm and 330 nm, respectively (Figure 5). The peak area ratio was close to 1∶1 for the two fluorophores. The previous mutational analysis indicates that the fluorescence emission is significantly quenched for the W69-only and W157-only mutants, and the emission maximum is centered at 327 nm and 318 nm for the W43-only and W131-only mutants, respectively [47]. Thus it seems that in the fluorescence of native γD-crystallin, the Class A & S fluorophore was mainly contributed by W131, and the Class I fluorophore was mainly by W43. The existence of a minor content of Class II fluorophores centered at 340 nm may arise from the fast exchange of protein conformations in solutions. At 75°C where the intermediate state appeared during the thermal unfolding of the WT γD-crystallin, the contribution of the Class A & S fluorophore decreased, while that of the Class I fluorophore increased correspondingly. This may result from an alteration in the microenvironments of W131 in the intermediate state or an additional contribution by the Trp residues that are quenched in the native state. At temperatures above 75°C, the contents of both the Class A & S and Class I fluorophores decreased, while that of Class II increased continuously. This suggested that the microenvironments of all Trp residues were changed to the more solvent-accessible state upon heating, which is confirmed by the simultaneous increase in the hydrophobic exposure as monitored by ANS fluorescence (Figure 3C). Similar results were obtained for the mutated γD-crystallin except that the transitions occurred at a relatively lower temperature when compared with the WT γD-crystallin.

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