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

PURPOSE: The COOH-terminal extension segment of αB-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 chapero...

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Autores principales: Liao, Jiahn-Haur, Lee, Jiahn-Shing, Wu, Shih-Hsiung, Chiou, Shyh-Horng
Formato: Texto
Lenguaje:English
Publicado: Molecular Vision 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2716931/
https://www.ncbi.nlm.nih.gov/pubmed/19641632
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author Liao, Jiahn-Haur
Lee, Jiahn-Shing
Wu, Shih-Hsiung
Chiou, Shyh-Horng
author_facet Liao, Jiahn-Haur
Lee, Jiahn-Shing
Wu, Shih-Hsiung
Chiou, Shyh-Horng
author_sort Liao, Jiahn-Haur
collection PubMed
description PURPOSE: The COOH-terminal extension segment of αB-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 αB-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 αB-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 αB-crystallin. The deletion of 12 residues from the COOH-terminal end greatly reduced the solubility, thermostability, and chaperone activity of αB-crystallin. On the contrary, the truncation of only 10 residues or less resulted in increased thermostability and enhanced anti-aggregation chaperone activity of αB-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 αB-crystallin.
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spelling pubmed-27169312009-07-28 COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine αB-crystallin Liao, Jiahn-Haur Lee, Jiahn-Shing Wu, Shih-Hsiung Chiou, Shyh-Horng Mol Vis Research Article PURPOSE: The COOH-terminal extension segment of αB-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 αB-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 αB-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 αB-crystallin. The deletion of 12 residues from the COOH-terminal end greatly reduced the solubility, thermostability, and chaperone activity of αB-crystallin. On the contrary, the truncation of only 10 residues or less resulted in increased thermostability and enhanced anti-aggregation chaperone activity of αB-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 αB-crystallin. Molecular Vision 2009-07-28 /pmc/articles/PMC2716931/ /pubmed/19641632 Text en http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Liao, Jiahn-Haur
Lee, Jiahn-Shing
Wu, Shih-Hsiung
Chiou, Shyh-Horng
COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine αB-crystallin
title COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine αB-crystallin
title_full COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine αB-crystallin
title_fullStr COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine αB-crystallin
title_full_unstemmed COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine αB-crystallin
title_short COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine αB-crystallin
title_sort cooh-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine αb-crystallin
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2716931/
https://www.ncbi.nlm.nih.gov/pubmed/19641632
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