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Structural analysis of the mutant protein D26G of human γS-crystallin, associated with Coppock cataract.

Karri S, Kasetti RB, Vendra VP, Chandani S, Balasubramanian D - Mol. Vis. (2013)

Bottom Line: The mutant also self-aggregated more readily (it turned turbid upon standing; at 65 °C, it started precipitating beyond 200 s, while the WT did not, even after 900 s).Molecular modeling showed that the Asp26-Arg84 contact (and the related Arg84-Asn54 interaction) was disturbed in the mutant, making the latter less compact around the mutation site.The cataract-associated mutant D26G of HGSC is remarkably close to the WT molecule in structural features, with only a microenvironmental change in the packing around the mutation site.

View Article: PubMed Central - PubMed

Affiliation: Prof. Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India.

ABSTRACT

Purpose: To analyze the protein structural features responsible for the aggregation properties of the mutant protein D26G human γS-crystallin (HGSC) associated with congenital Coppock-type cataract.

Methods: cDNAs of wild-type (WT) and D26G mutant HGSC were cloned and expressed in BL21 (DE3) pLysS cells and the proteins isolated and purified. Their secondary and tertiary structural features, aggregation tendencies, and structural stabilities were compared using spectroscopic (circular dichroism, intrinsic and extrinsic fluorescence), molecular modeling, and dynamics methods.

Results: No difference was observed between the conformational (secondary and tertiary structural) features and aggregation properties between the WT and D26G proteins. The mutant, however, was structurally less stable; it denatured at a slightly lower concentration of the added chemical denaturant (at 2.05 M guanidinium chloride, cf. 2.20 M for the WT) and at a slightly lower temperature (at 70.8 °C, cf. 72.0 °C for the WT). The mutant also self-aggregated more readily (it turned turbid upon standing; at 65 °C, it started precipitating beyond 200 s, while the WT did not, even after 900 s). Molecular modeling showed that the Asp26-Arg84 contact (and the related Arg84-Asn54 interaction) was disturbed in the mutant, making the latter less compact around the mutation site.

Conclusions: The cataract-associated mutant D26G of HGSC is remarkably close to the WT molecule in structural features, with only a microenvironmental change in the packing around the mutation site. This alteration appears sufficient to promote self-aggregation, resulting in peripheral cataract.

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Secondary and tertiary structural analysis of the proteins. A: The backbone folding does not alter upon mutation. Estimation of the secondary structures of the proteins, using far-ultraviolet circular dichroism (CD) spectra in the region 195–250 nm, using a JASCO CD spectrometer, at ambient temperature (27 °C), recorded with 2 mm path length cells. B: Intrinsic fluorescence of the wild-type and D26G human gamma S-crystallin (HGSC) differ little: Curve A (Blue): γS wild-type; B (Red): D26G; with excitation wavelength 295 nm and emission wavelength recorded from 300 to 400 nm, at ambient temperature, using a Hitachi spectrofluorimeter. C: The mutation has a slightly more set of surface-exposed residues: Extrinsic emission spectra of the surface probe Nile Red. A (Blue): γS wild-type; B (Red): D26G; extrinsic fluorescence spectra were recorded between 570 and 700 nm with excitation at 540 nm; and slit size 10 nm for excitation and emission. In each set of spectra in A, B, and C, the protein concentrations used were 5 µM (0.1 mg/ml) in 50 mM Tris buffer (pH 7.3), cell path length 3 mm, and spectra recorded at 5.0 nm excitation and emission slits. Spectra shown were the average of three runs.
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f1: Secondary and tertiary structural analysis of the proteins. A: The backbone folding does not alter upon mutation. Estimation of the secondary structures of the proteins, using far-ultraviolet circular dichroism (CD) spectra in the region 195–250 nm, using a JASCO CD spectrometer, at ambient temperature (27 °C), recorded with 2 mm path length cells. B: Intrinsic fluorescence of the wild-type and D26G human gamma S-crystallin (HGSC) differ little: Curve A (Blue): γS wild-type; B (Red): D26G; with excitation wavelength 295 nm and emission wavelength recorded from 300 to 400 nm, at ambient temperature, using a Hitachi spectrofluorimeter. C: The mutation has a slightly more set of surface-exposed residues: Extrinsic emission spectra of the surface probe Nile Red. A (Blue): γS wild-type; B (Red): D26G; extrinsic fluorescence spectra were recorded between 570 and 700 nm with excitation at 540 nm; and slit size 10 nm for excitation and emission. In each set of spectra in A, B, and C, the protein concentrations used were 5 µM (0.1 mg/ml) in 50 mM Tris buffer (pH 7.3), cell path length 3 mm, and spectra recorded at 5.0 nm excitation and emission slits. Spectra shown were the average of three runs.

Mentions: In Figure 1A, the secondary structural features of WT and D26G HGSC are compared using CD spectroscopy. The near identity of the spectra suggests that no significant changes occurred to the backbone conformation to the protein after the aspartic acid residue was substituted by glycine in the first Greek key motif. Both molecules are folded predominantly in the β-pleated sheet conformation, as shown by the negative band at 218 nm. Figure 1B shows the intrinsic fluorescence due to the tryptophan (and tyrosine) residues in the proteins. Emission in the 327 nm region suggests that the immediate microenvironment around the aromatic side chains is about the same, and the value of 327 nm suggests that they are buried in a nonpolar environment. We next attempted to quench this fluorescence using the ionic quencher KI, which accesses the surface regions of the protein [14]. The Stern-Volmer quenching constant for KI in the wild-type HGSC was estimated to be Ksv=0.12±0.02, while that for D26G was 0.21±0.04, suggesting that the mutation affects the microenvironment around the aromatic residues to become slightly more accessible. To check this further, we studied the binding of the proteins to the neutral extrinsic fluorescence probe Nile Red, which reports on the surface regions of the macromolecule [15]. Figure 1C shows the emission intensity of Nile Red bound to the mutant is progressively higher than when it is bound to WT; at 100 µM, the probe displays a higher intensity when bound to D26G than to WT (2.75 arbitrary units cf. 1.9). Likewise, when the other surface probe bis-ANS was used [16], it displayed an intensity of 12.5 units when bound to D26G and 10 units when bound to WT (figure not shown).


Structural analysis of the mutant protein D26G of human γS-crystallin, associated with Coppock cataract.

Karri S, Kasetti RB, Vendra VP, Chandani S, Balasubramanian D - Mol. Vis. (2013)

Secondary and tertiary structural analysis of the proteins. A: The backbone folding does not alter upon mutation. Estimation of the secondary structures of the proteins, using far-ultraviolet circular dichroism (CD) spectra in the region 195–250 nm, using a JASCO CD spectrometer, at ambient temperature (27 °C), recorded with 2 mm path length cells. B: Intrinsic fluorescence of the wild-type and D26G human gamma S-crystallin (HGSC) differ little: Curve A (Blue): γS wild-type; B (Red): D26G; with excitation wavelength 295 nm and emission wavelength recorded from 300 to 400 nm, at ambient temperature, using a Hitachi spectrofluorimeter. C: The mutation has a slightly more set of surface-exposed residues: Extrinsic emission spectra of the surface probe Nile Red. A (Blue): γS wild-type; B (Red): D26G; extrinsic fluorescence spectra were recorded between 570 and 700 nm with excitation at 540 nm; and slit size 10 nm for excitation and emission. In each set of spectra in A, B, and C, the protein concentrations used were 5 µM (0.1 mg/ml) in 50 mM Tris buffer (pH 7.3), cell path length 3 mm, and spectra recorded at 5.0 nm excitation and emission slits. Spectra shown were the average of three runs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675056&req=5

f1: Secondary and tertiary structural analysis of the proteins. A: The backbone folding does not alter upon mutation. Estimation of the secondary structures of the proteins, using far-ultraviolet circular dichroism (CD) spectra in the region 195–250 nm, using a JASCO CD spectrometer, at ambient temperature (27 °C), recorded with 2 mm path length cells. B: Intrinsic fluorescence of the wild-type and D26G human gamma S-crystallin (HGSC) differ little: Curve A (Blue): γS wild-type; B (Red): D26G; with excitation wavelength 295 nm and emission wavelength recorded from 300 to 400 nm, at ambient temperature, using a Hitachi spectrofluorimeter. C: The mutation has a slightly more set of surface-exposed residues: Extrinsic emission spectra of the surface probe Nile Red. A (Blue): γS wild-type; B (Red): D26G; extrinsic fluorescence spectra were recorded between 570 and 700 nm with excitation at 540 nm; and slit size 10 nm for excitation and emission. In each set of spectra in A, B, and C, the protein concentrations used were 5 µM (0.1 mg/ml) in 50 mM Tris buffer (pH 7.3), cell path length 3 mm, and spectra recorded at 5.0 nm excitation and emission slits. Spectra shown were the average of three runs.
Mentions: In Figure 1A, the secondary structural features of WT and D26G HGSC are compared using CD spectroscopy. The near identity of the spectra suggests that no significant changes occurred to the backbone conformation to the protein after the aspartic acid residue was substituted by glycine in the first Greek key motif. Both molecules are folded predominantly in the β-pleated sheet conformation, as shown by the negative band at 218 nm. Figure 1B shows the intrinsic fluorescence due to the tryptophan (and tyrosine) residues in the proteins. Emission in the 327 nm region suggests that the immediate microenvironment around the aromatic side chains is about the same, and the value of 327 nm suggests that they are buried in a nonpolar environment. We next attempted to quench this fluorescence using the ionic quencher KI, which accesses the surface regions of the protein [14]. The Stern-Volmer quenching constant for KI in the wild-type HGSC was estimated to be Ksv=0.12±0.02, while that for D26G was 0.21±0.04, suggesting that the mutation affects the microenvironment around the aromatic residues to become slightly more accessible. To check this further, we studied the binding of the proteins to the neutral extrinsic fluorescence probe Nile Red, which reports on the surface regions of the macromolecule [15]. Figure 1C shows the emission intensity of Nile Red bound to the mutant is progressively higher than when it is bound to WT; at 100 µM, the probe displays a higher intensity when bound to D26G than to WT (2.75 arbitrary units cf. 1.9). Likewise, when the other surface probe bis-ANS was used [16], it displayed an intensity of 12.5 units when bound to D26G and 10 units when bound to WT (figure not shown).

Bottom Line: The mutant also self-aggregated more readily (it turned turbid upon standing; at 65 °C, it started precipitating beyond 200 s, while the WT did not, even after 900 s).Molecular modeling showed that the Asp26-Arg84 contact (and the related Arg84-Asn54 interaction) was disturbed in the mutant, making the latter less compact around the mutation site.The cataract-associated mutant D26G of HGSC is remarkably close to the WT molecule in structural features, with only a microenvironmental change in the packing around the mutation site.

View Article: PubMed Central - PubMed

Affiliation: Prof. Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India.

ABSTRACT

Purpose: To analyze the protein structural features responsible for the aggregation properties of the mutant protein D26G human γS-crystallin (HGSC) associated with congenital Coppock-type cataract.

Methods: cDNAs of wild-type (WT) and D26G mutant HGSC were cloned and expressed in BL21 (DE3) pLysS cells and the proteins isolated and purified. Their secondary and tertiary structural features, aggregation tendencies, and structural stabilities were compared using spectroscopic (circular dichroism, intrinsic and extrinsic fluorescence), molecular modeling, and dynamics methods.

Results: No difference was observed between the conformational (secondary and tertiary structural) features and aggregation properties between the WT and D26G proteins. The mutant, however, was structurally less stable; it denatured at a slightly lower concentration of the added chemical denaturant (at 2.05 M guanidinium chloride, cf. 2.20 M for the WT) and at a slightly lower temperature (at 70.8 °C, cf. 72.0 °C for the WT). The mutant also self-aggregated more readily (it turned turbid upon standing; at 65 °C, it started precipitating beyond 200 s, while the WT did not, even after 900 s). Molecular modeling showed that the Asp26-Arg84 contact (and the related Arg84-Asn54 interaction) was disturbed in the mutant, making the latter less compact around the mutation site.

Conclusions: The cataract-associated mutant D26G of HGSC is remarkably close to the WT molecule in structural features, with only a microenvironmental change in the packing around the mutation site. This alteration appears sufficient to promote self-aggregation, resulting in peripheral cataract.

Show MeSH
Related in: MedlinePlus