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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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Effect of the G61C mutation on γD-crystallin structure.(A) SEC profile of the WT and mutated γD-crystallin. (B) Far-UV CD spectra of the native proteins and the denatured states in 8 M urea or 6 M GdnHCl. (C) Intrinsic fluorescence spectra of the native proteins and denatured states in 8 M urea or 6 M GdnHCl. The excitation wavelength of the intrinsic fluorescence was 295 nm. (D) ANS fluorescence spectra of the WT and mutated γD-crystallin. The excitation wavelength of the ANS fluorescence was 380 nm. The protein samples were prepared by dissolving the proteins with a final protein concentration of 0.2 mg/ml in 10 mM PBS buffer, pH 7.0, containing 1 mM EDTA and 2 mM DTT.
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pone-0020564-g002: Effect of the G61C mutation on γD-crystallin structure.(A) SEC profile of the WT and mutated γD-crystallin. (B) Far-UV CD spectra of the native proteins and the denatured states in 8 M urea or 6 M GdnHCl. (C) Intrinsic fluorescence spectra of the native proteins and denatured states in 8 M urea or 6 M GdnHCl. The excitation wavelength of the intrinsic fluorescence was 295 nm. (D) ANS fluorescence spectra of the WT and mutated γD-crystallin. The excitation wavelength of the ANS fluorescence was 380 nm. The protein samples were prepared by dissolving the proteins with a final protein concentration of 0.2 mg/ml in 10 mM PBS buffer, pH 7.0, containing 1 mM EDTA and 2 mM DTT.

Mentions: The purified proteins were found to be homogenous as evaluated by SDS-PAGE and SEC analysis. In the SEC profile, both the WT and mutated proteins eluted as a single peak and the elusion volume was not significantly affected by mutation (Figure 2A). This suggested that the mutation did not affect the oligomeric state or the shape of the γD-crystallin molecule. The effect of the mutation on γD-crystallin structure was investigated via spectroscopic methods (Figure 2B–2D). The CD spectrum of the WT γD-crystallin showed a single negative peak at around 217 nm, revealing that the major regular secondary structure content was β-sheet. This observation is quite consistent with the crystal structure of γD-crystallin [34], which composes four Greek-key β-sheet divided into two domains (Figure 1). The Trp fluorescence of the WT protein was centered at around 325 nm, suggesting that the Trp residues of γD-crystallin mainly located in hydrophobic microenvironments. The hydrophobic exposure of the proteins were evaluated by ANS fluorescence, and no difference was found between the WT and mutated γD-crystallins. The almost superimposed spectra of the WT and mutated proteins shown in Figure 2 clearly indicated that the mutation did not significantly affect the secondary and tertiary structures of γD-crystallin. Both the WT and mutated proteins could maintain most of their native structures in 8 M urea, which can efficiently denature many other proteins [44], suggesting that both proteins were resistant to urea-induced denaturation. In 6 M GdnHCl, both proteins were fully unfolded as revealed by the lack of ordered secondary structures (Figure 2B) and the fully water-exposed Trp residues (Figure 2C).


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 γD-crystallin structure.(A) SEC profile of the WT and mutated γD-crystallin. (B) Far-UV CD spectra of the native proteins and the denatured states in 8 M urea or 6 M GdnHCl. (C) Intrinsic fluorescence spectra of the native proteins and denatured states in 8 M urea or 6 M GdnHCl. The excitation wavelength of the intrinsic fluorescence was 295 nm. (D) ANS fluorescence spectra of the WT and mutated γD-crystallin. The excitation wavelength of the ANS fluorescence was 380 nm. The protein samples were prepared by dissolving the proteins with a final protein concentration of 0.2 mg/ml in 10 mM PBS buffer, pH 7.0, containing 1 mM EDTA and 2 mM DTT.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020564-g002: Effect of the G61C mutation on γD-crystallin structure.(A) SEC profile of the WT and mutated γD-crystallin. (B) Far-UV CD spectra of the native proteins and the denatured states in 8 M urea or 6 M GdnHCl. (C) Intrinsic fluorescence spectra of the native proteins and denatured states in 8 M urea or 6 M GdnHCl. The excitation wavelength of the intrinsic fluorescence was 295 nm. (D) ANS fluorescence spectra of the WT and mutated γD-crystallin. The excitation wavelength of the ANS fluorescence was 380 nm. The protein samples were prepared by dissolving the proteins with a final protein concentration of 0.2 mg/ml in 10 mM PBS buffer, pH 7.0, containing 1 mM EDTA and 2 mM DTT.
Mentions: The purified proteins were found to be homogenous as evaluated by SDS-PAGE and SEC analysis. In the SEC profile, both the WT and mutated proteins eluted as a single peak and the elusion volume was not significantly affected by mutation (Figure 2A). This suggested that the mutation did not affect the oligomeric state or the shape of the γD-crystallin molecule. The effect of the mutation on γD-crystallin structure was investigated via spectroscopic methods (Figure 2B–2D). The CD spectrum of the WT γD-crystallin showed a single negative peak at around 217 nm, revealing that the major regular secondary structure content was β-sheet. This observation is quite consistent with the crystal structure of γD-crystallin [34], which composes four Greek-key β-sheet divided into two domains (Figure 1). The Trp fluorescence of the WT protein was centered at around 325 nm, suggesting that the Trp residues of γD-crystallin mainly located in hydrophobic microenvironments. The hydrophobic exposure of the proteins were evaluated by ANS fluorescence, and no difference was found between the WT and mutated γD-crystallins. The almost superimposed spectra of the WT and mutated proteins shown in Figure 2 clearly indicated that the mutation did not significantly affect the secondary and tertiary structures of γD-crystallin. Both the WT and mutated proteins could maintain most of their native structures in 8 M urea, which can efficiently denature many other proteins [44], suggesting that both proteins were resistant to urea-induced denaturation. In 6 M GdnHCl, both proteins were fully unfolded as revealed by the lack of ordered secondary structures (Figure 2B) and the fully water-exposed Trp residues (Figure 2C).

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