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COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine alphaB-crystallin.

Liao JH, Lee JS, Wu SH, Chiou SH - Mol. Vis. (2009)

Bottom Line: The deletion of 12 residues from the COOH-terminal end greatly reduced the solubility, thermostability, and chaperone activity of alphaB-crystallin.On the contrary, the truncation of only 10 residues or less resulted in increased thermostability and enhanced anti-aggregation chaperone activity of alphaB-crystallin, with a maximal effect occurring on elimination of the last two residues.Our study clearly demonstrated that both the length and electrostatic charge of the COOH-terminal segment play crucial roles in governing the structural stability and chaperone activity of alphaB-crystallin.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

ABSTRACT

Purpose: The COOH-terminal extension segment of alphaB-crystallin, a member of small heat shock protein (sHSP) family, appears to be a flexible polypeptide segment susceptible to proteolytic truncation and modifications under physiological conditions. To investigate its role on the structure and chaperone-like activity, we constructed various mutants of porcine alphaB-crystallin with either COOH-terminal serial truncations or site-specific mutagenesis on the last two residues.

Methods: The structures of these mutants were analyzed by circular dichroism (CD) spectroscopy, fluorescence spectra, mass spectrometry, Gel-permeation FPLC, and dynamic light-scattering spectrophotometry. Chaperone activity assays were performed under thermal and non-thermal stresses. The stability of proteins was examined by turbidity assays and CD spectra.

Results: All mutants showed similar secondary and tertiary structural features to the wild-type alphaB-crystallin as revealed by circular dichroism. However, truncations of the COOH-terminal segment generated crystallin aggregates with a molecular size slightly smaller than that of the wild-type alphaB-crystallin. The deletion of 12 residues from the COOH-terminal end greatly reduced the solubility, thermostability, and chaperone activity of alphaB-crystallin. On the contrary, the truncation of only 10 residues or less resulted in increased thermostability and enhanced anti-aggregation chaperone activity of alphaB-crystallin, with a maximal effect occurring on elimination of the last two residues. Moreover, displacing the last two lysines with glutamates or other neutral amino acids tended to show even higher chaperone activity than the deletion mutants.

Conclusions: Our study clearly demonstrated that both the length and electrostatic charge of the COOH-terminal segment play crucial roles in governing the structural stability and chaperone activity of alphaB-crystallin.

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Related in: MedlinePlus

The COOH-terminal sequences of αB-crystallin and its mutants. A: The amino acid sequences from Q151 to the COOH-terminal of wild-type (WT) αB-crystallin and its truncated mutants. Various truncated mutants of porcine αB-crystallin have been constructed in order to study the functions of the COOH-terminal extension fragment of αB-crystallin. The amino acid (E164) of these mutants is underlined and marked with an asterisk. B: The amino acid sequences from I160 to the COOH-terminus of wild-type αB-crystallin and its mutants. Various mutants were constructed to study effects of amino-acid substitutions at position 174 and 175. The amino acid (P173) of these mutants is underlined and marked with an asterisk.
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f1: The COOH-terminal sequences of αB-crystallin and its mutants. A: The amino acid sequences from Q151 to the COOH-terminal of wild-type (WT) αB-crystallin and its truncated mutants. Various truncated mutants of porcine αB-crystallin have been constructed in order to study the functions of the COOH-terminal extension fragment of αB-crystallin. The amino acid (E164) of these mutants is underlined and marked with an asterisk. B: The amino acid sequences from I160 to the COOH-terminus of wild-type αB-crystallin and its mutants. Various mutants were constructed to study effects of amino-acid substitutions at position 174 and 175. The amino acid (P173) of these mutants is underlined and marked with an asterisk.

Mentions: Various truncated mutants of porcine αB-crystallin have been constructed to study the function of COOH-terminal extension segment of αB-crystallin (Figure 1A). These constructs were transformed in E. coli strain BL21 (DE3) and overexpressed in the presence of IPTG. Soluble recombinant proteins were purified by TSK HW-55 gel filtration, followed by reverse-phase HPLC (C4). Recombinant αB-crystallin and mutants were dissolved in 8 M urea, and refolded by eluting in a column of TSK HW-55(F). The recombinant αB-crystallin and mutants were thus reassociated and refolded after removal of urea. The purity of these proteins was checked by SDS-PAGE (Figure 2A). The deletion mutants Δ2, Δ5, and Δ7 show similar solubility with recombinant αB-crystallin. The Δ10 mutant showed a lower solubility than wild-type αB-crystallin when measured at low temperature; however, the Δ12 mutant showed even worse solubility and stability under ambient temperature. We also constructed various mutants by replacing amino acids to study the effects of COOH-terminal lysines (Figure 1B). The purity of these proteins was also checked and confirmed by SDS-PAGE (Figure 2B,C). The pI values of recombinant αB-crystallin and mutants were shown in Figure 3A,B. The approximate pH of the isoelectric-focusing gel was estimated from pI calibration kits. The pI value of the recombinant αB-crystallin corresponded to about that of bovine carbonic anhydrase B (pI 5.85). All mutants show pI values locating between 5.85 (bovine carbonic anhydrase B) and 6.55 (human carbonic anhydrase B). Recombinant αB-crystallin and mutants show distinct focusing zones with broad distribution of pI instead of sharp bands characteristic of pI marker proteins. The anomaly of broad distribution of pI values for recombinant αB-crystallin and its mutants may be due to their inherent polydisperse properties in their surface charges, and the existence of a rapid equilibrium of association-dissociation behavior associated with crystallin aggregation during electrophoresis.


COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine alphaB-crystallin.

Liao JH, Lee JS, Wu SH, Chiou SH - Mol. Vis. (2009)

The COOH-terminal sequences of αB-crystallin and its mutants. A: The amino acid sequences from Q151 to the COOH-terminal of wild-type (WT) αB-crystallin and its truncated mutants. Various truncated mutants of porcine αB-crystallin have been constructed in order to study the functions of the COOH-terminal extension fragment of αB-crystallin. The amino acid (E164) of these mutants is underlined and marked with an asterisk. B: The amino acid sequences from I160 to the COOH-terminus of wild-type αB-crystallin and its mutants. Various mutants were constructed to study effects of amino-acid substitutions at position 174 and 175. The amino acid (P173) of these mutants is underlined and marked with an asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2716931&req=5

f1: The COOH-terminal sequences of αB-crystallin and its mutants. A: The amino acid sequences from Q151 to the COOH-terminal of wild-type (WT) αB-crystallin and its truncated mutants. Various truncated mutants of porcine αB-crystallin have been constructed in order to study the functions of the COOH-terminal extension fragment of αB-crystallin. The amino acid (E164) of these mutants is underlined and marked with an asterisk. B: The amino acid sequences from I160 to the COOH-terminus of wild-type αB-crystallin and its mutants. Various mutants were constructed to study effects of amino-acid substitutions at position 174 and 175. The amino acid (P173) of these mutants is underlined and marked with an asterisk.
Mentions: Various truncated mutants of porcine αB-crystallin have been constructed to study the function of COOH-terminal extension segment of αB-crystallin (Figure 1A). These constructs were transformed in E. coli strain BL21 (DE3) and overexpressed in the presence of IPTG. Soluble recombinant proteins were purified by TSK HW-55 gel filtration, followed by reverse-phase HPLC (C4). Recombinant αB-crystallin and mutants were dissolved in 8 M urea, and refolded by eluting in a column of TSK HW-55(F). The recombinant αB-crystallin and mutants were thus reassociated and refolded after removal of urea. The purity of these proteins was checked by SDS-PAGE (Figure 2A). The deletion mutants Δ2, Δ5, and Δ7 show similar solubility with recombinant αB-crystallin. The Δ10 mutant showed a lower solubility than wild-type αB-crystallin when measured at low temperature; however, the Δ12 mutant showed even worse solubility and stability under ambient temperature. We also constructed various mutants by replacing amino acids to study the effects of COOH-terminal lysines (Figure 1B). The purity of these proteins was also checked and confirmed by SDS-PAGE (Figure 2B,C). The pI values of recombinant αB-crystallin and mutants were shown in Figure 3A,B. The approximate pH of the isoelectric-focusing gel was estimated from pI calibration kits. The pI value of the recombinant αB-crystallin corresponded to about that of bovine carbonic anhydrase B (pI 5.85). All mutants show pI values locating between 5.85 (bovine carbonic anhydrase B) and 6.55 (human carbonic anhydrase B). Recombinant αB-crystallin and mutants show distinct focusing zones with broad distribution of pI instead of sharp bands characteristic of pI marker proteins. The anomaly of broad distribution of pI values for recombinant αB-crystallin and its mutants may be due to their inherent polydisperse properties in their surface charges, and the existence of a rapid equilibrium of association-dissociation behavior associated with crystallin aggregation during electrophoresis.

Bottom Line: The deletion of 12 residues from the COOH-terminal end greatly reduced the solubility, thermostability, and chaperone activity of alphaB-crystallin.On the contrary, the truncation of only 10 residues or less resulted in increased thermostability and enhanced anti-aggregation chaperone activity of alphaB-crystallin, with a maximal effect occurring on elimination of the last two residues.Our study clearly demonstrated that both the length and electrostatic charge of the COOH-terminal segment play crucial roles in governing the structural stability and chaperone activity of alphaB-crystallin.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

ABSTRACT

Purpose: The COOH-terminal extension segment of alphaB-crystallin, a member of small heat shock protein (sHSP) family, appears to be a flexible polypeptide segment susceptible to proteolytic truncation and modifications under physiological conditions. To investigate its role on the structure and chaperone-like activity, we constructed various mutants of porcine alphaB-crystallin with either COOH-terminal serial truncations or site-specific mutagenesis on the last two residues.

Methods: The structures of these mutants were analyzed by circular dichroism (CD) spectroscopy, fluorescence spectra, mass spectrometry, Gel-permeation FPLC, and dynamic light-scattering spectrophotometry. Chaperone activity assays were performed under thermal and non-thermal stresses. The stability of proteins was examined by turbidity assays and CD spectra.

Results: All mutants showed similar secondary and tertiary structural features to the wild-type alphaB-crystallin as revealed by circular dichroism. However, truncations of the COOH-terminal segment generated crystallin aggregates with a molecular size slightly smaller than that of the wild-type alphaB-crystallin. The deletion of 12 residues from the COOH-terminal end greatly reduced the solubility, thermostability, and chaperone activity of alphaB-crystallin. On the contrary, the truncation of only 10 residues or less resulted in increased thermostability and enhanced anti-aggregation chaperone activity of alphaB-crystallin, with a maximal effect occurring on elimination of the last two residues. Moreover, displacing the last two lysines with glutamates or other neutral amino acids tended to show even higher chaperone activity than the deletion mutants.

Conclusions: Our study clearly demonstrated that both the length and electrostatic charge of the COOH-terminal segment play crucial roles in governing the structural stability and chaperone activity of alphaB-crystallin.

Show MeSH
Related in: MedlinePlus