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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 thermal stability evaluated by the ellipticity at 222 nm in the far-UV CD spectra (A), the maximum emission wavelength (Emax) of the intrinsic fluorescence (B), the intensity at 470 nm of the ANS fluorescence (C) and the resonance Rayleigh light scattering excited at 295 nm (D).The temperature was controlled by a water bath, and the data were recorded at a given temperature after a 2-min equilibration. The raw data were fitted to a two-state model, and the fitting results are shown by solid lines.
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pone-0020564-g003: Effect of the G61C mutation on γD-crystallin thermal stability evaluated by the ellipticity at 222 nm in the far-UV CD spectra (A), the maximum emission wavelength (Emax) of the intrinsic fluorescence (B), the intensity at 470 nm of the ANS fluorescence (C) and the resonance Rayleigh light scattering excited at 295 nm (D).The temperature was controlled by a water bath, and the data were recorded at a given temperature after a 2-min equilibration. The raw data were fitted to a two-state model, and the fitting results are shown by solid lines.

Mentions: The effect of the G61 C mutation on γD-crystallin thermal stability was investigated by increasing the incubation temperature every 2°C from 25°C to 85°C. The structural changes were monitored by far-UV CD, intrinsic fluorescence, ANS fluorescence and resonance Raleigh light scattering, which reflects the secondary structure, tertiary structure or microenvironment of the Trp residues, hydrophobic exposure and oligomeric state of proteins. As presented in Figure 3, all transition curves exhibited an apparent two-state process. It is worth noting that no aggregation was detected when the temperature is below 78°C for G61C and 82°C for the WT γD-crystallin, and the changes in the resonance Raleigh light scattering mainly reflect the occurrence of non-native soluble oligomers [38]. The midpoint temperature where half of the unfolding occurred (T0.5) of the mutated protein was ∼74°C when probed by far-UV CD, intrinsic fluorescence and light scattering. The T0.5 value of the WT protein was above 80°C, while the exact value could not be obtained for the data from intrinsic fluorescence and light scattering since the transition curves did not reach its equilibrium. ANS, a fluorescent molecule with the ability to specifically bind with the hydrophobic site [45], is a frequently used probe to detect the hydrophobic exposure of proteins. The T0.5 value from ANS fluorescence was ∼4°C lower than that from the other three probes for both proteins. Thus the inconsistency of the transition curves suggested that the thermal denaturation of both the WT and mutated γD-crystallin might involve sequential events, and the exposure of the hydrophobic core was an early event during γD-crystallin thermal unfolding as revealed by the ANS fluorescence.


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 thermal stability evaluated by the ellipticity at 222 nm in the far-UV CD spectra (A), the maximum emission wavelength (Emax) of the intrinsic fluorescence (B), the intensity at 470 nm of the ANS fluorescence (C) and the resonance Rayleigh light scattering excited at 295 nm (D).The temperature was controlled by a water bath, and the data were recorded at a given temperature after a 2-min equilibration. The raw data were fitted to a two-state model, and the fitting results are shown by solid lines.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020564-g003: Effect of the G61C mutation on γD-crystallin thermal stability evaluated by the ellipticity at 222 nm in the far-UV CD spectra (A), the maximum emission wavelength (Emax) of the intrinsic fluorescence (B), the intensity at 470 nm of the ANS fluorescence (C) and the resonance Rayleigh light scattering excited at 295 nm (D).The temperature was controlled by a water bath, and the data were recorded at a given temperature after a 2-min equilibration. The raw data were fitted to a two-state model, and the fitting results are shown by solid lines.
Mentions: The effect of the G61 C mutation on γD-crystallin thermal stability was investigated by increasing the incubation temperature every 2°C from 25°C to 85°C. The structural changes were monitored by far-UV CD, intrinsic fluorescence, ANS fluorescence and resonance Raleigh light scattering, which reflects the secondary structure, tertiary structure or microenvironment of the Trp residues, hydrophobic exposure and oligomeric state of proteins. As presented in Figure 3, all transition curves exhibited an apparent two-state process. It is worth noting that no aggregation was detected when the temperature is below 78°C for G61C and 82°C for the WT γD-crystallin, and the changes in the resonance Raleigh light scattering mainly reflect the occurrence of non-native soluble oligomers [38]. The midpoint temperature where half of the unfolding occurred (T0.5) of the mutated protein was ∼74°C when probed by far-UV CD, intrinsic fluorescence and light scattering. The T0.5 value of the WT protein was above 80°C, while the exact value could not be obtained for the data from intrinsic fluorescence and light scattering since the transition curves did not reach its equilibrium. ANS, a fluorescent molecule with the ability to specifically bind with the hydrophobic site [45], is a frequently used probe to detect the hydrophobic exposure of proteins. The T0.5 value from ANS fluorescence was ∼4°C lower than that from the other three probes for both proteins. Thus the inconsistency of the transition curves suggested that the thermal denaturation of both the WT and mutated γD-crystallin might involve sequential events, and the exposure of the hydrophobic core was an early event during γD-crystallin thermal unfolding as revealed by the ANS fluorescence.

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