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

[摘要] Purpose: The COOH-terminal extensionsegment of αB-crystallin, a member of small heat shock protein (sHSP)family, appears to be a flexible polypeptide segment susceptible toproteolytic truncation and modifications under physiologicalconditions. To investigate its role on the structure and chaperone-likeactivity, we constructed various mutants of porcine αB-crystallin witheither COOH-terminal serial truncations or site-specific mutagenesis onthe last two residues. Methods: The structures of these mutantswere analyzed by circular dichroism (CD) spectroscopy, fluorescencespectra, mass spectrometry, Gel-permeation FPLC, and dynamiclight-scattering spectrophotometry. Chaperone activity assays wereperformed under thermal and non-thermal stresses. The stability ofproteins was examined by turbidity assays and CD spectra. Results: All mutants showed similarsecondary and tertiary structural features to the wild-typeαB-crystallin as revealed by circular dichroism. However, truncationsof the COOH-terminal segment generated crystallin aggregates with amolecular size slightly smaller than that of the wild-typeαB-crystallin. The deletion of 12 residues from the COOH-terminal endgreatly reduced the solubility, thermostability, and chaperone activityof αB-crystallin. On the contrary, the truncation of only 10 residuesor less resulted in increased thermostability and enhancedanti-aggregation chaperone activity of αB-crystallin, with a maximaleffect occurring on elimination of the last two residues. Moreover,displacing the last two lysines with glutamates or other neutral aminoacids tended to show even higher chaperone activity than the deletionmutants. Conclusions: Our study clearlydemonstrated that both the length and electrostatic charge of theCOOH-terminal segment play crucial roles in governing the structuralstability and chaperone activity of αB-crystallin.

[学科分类] 生物化学/生物物理