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Structural and aggregation behavior of the human γD-crystallin mutant E107A, associated with congenital nuclear cataract.

Vendra VP, Balasubramanian D - Mol. Vis. (2010)

Bottom Line: The mutant protein was found to be remarkably similar in its secondary and tertiary structural features to the wild type.Its structural stability, analyzed by guanidinium chloride-induced denaturation, was also found to be similar.E107A HGDC is yet another example of how a point mutation in the protein does not affect its conformation and stability but leads to substantial reduction in solubility and generation of light scattering aggregate particles in vitro and in situ when introduced into cell lines.

View Article: PubMed Central - PubMed

Affiliation: Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India.

ABSTRACT

Purpose: To analyze the conformational features and aggregation properties of the mutant protein E107A human γD-crystallin (HGDC), associated with congenital nuclear cataract.

Methods: cDNAs of wild type and E107A mutant were cloned and expressed in BL21 (DE3) pLysS cells and the proteins isolated and purified. The conformational properties and structural stability of the two proteins were compared using circular dichroism and fluorescence spectroscopic analysis. His-tagged cDNAs of the two proteins were transfected into HLE-3B human lens epithelial cells, and into HeLa cells and their in situ aggregation properties compared using immunofluorescence.

Results: The mutant protein was found to be remarkably similar in its secondary and tertiary structural features to the wild type. Its structural stability, analyzed by guanidinium chloride-induced denaturation, was also found to be similar. Its solubility, however, was over hundred-fold less than that of the wild type, and it had the tendency to precipitate and form light scattering particles. That it had the tendency to self- aggregate was noticed by using bis-ANS and Nile Red as extrinsic fluorescent probes. Such aggregation was also seen in situ when transfected and expressed in HLE-3B and in HeLa cell lines.

Conclusions: E107A HGDC is yet another example of how a point mutation in the protein does not affect its conformation and stability but leads to substantial reduction in solubility and generation of light scattering aggregate particles in vitro and in situ when introduced into cell lines.

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Related in: MedlinePlus

Wild type and E107A mutant differ very little in their surface exposure of apolar residues and in their structural stability. A: Intrinsic fluorescence of wild-type and E107A mutant HGDC. The protein concentrations used were 10 μM (0.2 mg/ml) in MOPS buffer, pH 7.3, cell path length 2 mm, and spectra were recorded at room temperature, using an excitation wavelength of 295 nm, with 2.5 nm slits. B: Guanidinium chloride–induced denaturation of wild-type and E107A HGDC. The relative emission intensity of the 360 nm band (of the denatured form) was compared to that of the 320 nm band (of the native protein) and monitored as a function of denaturant concentration.
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f2: Wild type and E107A mutant differ very little in their surface exposure of apolar residues and in their structural stability. A: Intrinsic fluorescence of wild-type and E107A mutant HGDC. The protein concentrations used were 10 μM (0.2 mg/ml) in MOPS buffer, pH 7.3, cell path length 2 mm, and spectra were recorded at room temperature, using an excitation wavelength of 295 nm, with 2.5 nm slits. B: Guanidinium chloride–induced denaturation of wild-type and E107A HGDC. The relative emission intensity of the 360 nm band (of the denatured form) was compared to that of the 320 nm band (of the native protein) and monitored as a function of denaturant concentration.

Mentions: Figure 1A compares the secondary structure of wild-type and E107A HGDC, using their far-UV circular dichroism spectra. The two curves are essentially superimposable, suggesting that the replacement of Glu by Ala in the molecule does not affect the backbone conformation of the protein chain in any significant manner. Figure 1B, which compares the near-UV CD spectra, reveals that the tertiary structure of the protein changes in a minor fashion (slight reduction in the residue molar ellipticity) upon the replacement of Glu by Ala. Figure 2A compares the intrinsic fluorescence spectra of the two molecules. Wild-type HGDC emits with a band maximum at 327.5 nm and intensity of 78.5 (arbitrary units), while E107A emits at 329.5 nm with an intensity of 76.2 units. Apparently, replacement of an anionic charge in the side chain of residue 107 by a nonpolar neutral moiety appears to have changed the microenvironment of the Trp and Tyr residues only in a minor manner.


Structural and aggregation behavior of the human γD-crystallin mutant E107A, associated with congenital nuclear cataract.

Vendra VP, Balasubramanian D - Mol. Vis. (2010)

Wild type and E107A mutant differ very little in their surface exposure of apolar residues and in their structural stability. A: Intrinsic fluorescence of wild-type and E107A mutant HGDC. The protein concentrations used were 10 μM (0.2 mg/ml) in MOPS buffer, pH 7.3, cell path length 2 mm, and spectra were recorded at room temperature, using an excitation wavelength of 295 nm, with 2.5 nm slits. B: Guanidinium chloride–induced denaturation of wild-type and E107A HGDC. The relative emission intensity of the 360 nm band (of the denatured form) was compared to that of the 320 nm band (of the native protein) and monitored as a function of denaturant concentration.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3008718&req=5

f2: Wild type and E107A mutant differ very little in their surface exposure of apolar residues and in their structural stability. A: Intrinsic fluorescence of wild-type and E107A mutant HGDC. The protein concentrations used were 10 μM (0.2 mg/ml) in MOPS buffer, pH 7.3, cell path length 2 mm, and spectra were recorded at room temperature, using an excitation wavelength of 295 nm, with 2.5 nm slits. B: Guanidinium chloride–induced denaturation of wild-type and E107A HGDC. The relative emission intensity of the 360 nm band (of the denatured form) was compared to that of the 320 nm band (of the native protein) and monitored as a function of denaturant concentration.
Mentions: Figure 1A compares the secondary structure of wild-type and E107A HGDC, using their far-UV circular dichroism spectra. The two curves are essentially superimposable, suggesting that the replacement of Glu by Ala in the molecule does not affect the backbone conformation of the protein chain in any significant manner. Figure 1B, which compares the near-UV CD spectra, reveals that the tertiary structure of the protein changes in a minor fashion (slight reduction in the residue molar ellipticity) upon the replacement of Glu by Ala. Figure 2A compares the intrinsic fluorescence spectra of the two molecules. Wild-type HGDC emits with a band maximum at 327.5 nm and intensity of 78.5 (arbitrary units), while E107A emits at 329.5 nm with an intensity of 76.2 units. Apparently, replacement of an anionic charge in the side chain of residue 107 by a nonpolar neutral moiety appears to have changed the microenvironment of the Trp and Tyr residues only in a minor manner.

Bottom Line: The mutant protein was found to be remarkably similar in its secondary and tertiary structural features to the wild type.Its structural stability, analyzed by guanidinium chloride-induced denaturation, was also found to be similar.E107A HGDC is yet another example of how a point mutation in the protein does not affect its conformation and stability but leads to substantial reduction in solubility and generation of light scattering aggregate particles in vitro and in situ when introduced into cell lines.

View Article: PubMed Central - PubMed

Affiliation: Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India.

ABSTRACT

Purpose: To analyze the conformational features and aggregation properties of the mutant protein E107A human γD-crystallin (HGDC), associated with congenital nuclear cataract.

Methods: cDNAs of wild type and E107A mutant were cloned and expressed in BL21 (DE3) pLysS cells and the proteins isolated and purified. The conformational properties and structural stability of the two proteins were compared using circular dichroism and fluorescence spectroscopic analysis. His-tagged cDNAs of the two proteins were transfected into HLE-3B human lens epithelial cells, and into HeLa cells and their in situ aggregation properties compared using immunofluorescence.

Results: The mutant protein was found to be remarkably similar in its secondary and tertiary structural features to the wild type. Its structural stability, analyzed by guanidinium chloride-induced denaturation, was also found to be similar. Its solubility, however, was over hundred-fold less than that of the wild type, and it had the tendency to precipitate and form light scattering particles. That it had the tendency to self- aggregate was noticed by using bis-ANS and Nile Red as extrinsic fluorescent probes. Such aggregation was also seen in situ when transfected and expressed in HLE-3B and in HeLa cell lines.

Conclusions: E107A HGDC is yet another example of how a point mutation in the protein does not affect its conformation and stability but leads to substantial reduction in solubility and generation of light scattering aggregate particles in vitro and in situ when introduced into cell lines.

Show MeSH
Related in: MedlinePlus