Dynamic Regulation of Microtubule Assembly by the Shsp and Molecular Chaperone, Human B Crystallin

Abstract

Purpose: : To evaluate the function of interactive sequences in human αB crystallin on dynamic subunit exchange and microtubule assembly.

Methods: : Five interactive sequences in αB crystallin were synthesized as individual peptides for experimental studies on the assembly of microtubules in vitro using a fluorescence DAPI assay (Ghosh et al. 2007 PloS one 6:3498). Mutations at corresponding positions in full-length αB crystallin were used experimentally to confirm the importance of these interactive sequences in the αB crystallin:tubulin interaction.

Results: : αB crystallin inhibited microtubule assembly at molar ratios >2:1, αB crystallin:tubulin and promoted microtubule assembly at molar ratios between 1:4 and 2:1. The plot of assembly versus molar ratio was parabolic with a maximum at approximately 0.5:1 αB:tubulin. The synthetic peptide corresponding to the αB crystallin interactive sequence, ₁₁₃FISREFHR₁₂₀, in the β3 strand of the α-crystallin core domain partially inhibited microtubule assembly. In contrast, the peptides ₁₃₁LTITSSLSSDGV₁₄₂ in the β8 strand and loop region of the core domain and ₁₅₆ERTIPITRE₁₆₄ in the C-terminal extension promoted microtubule assembly. Mutations in these exposed interactive sequences in full-length αB crystallin altered interactions with microtubules.

Keywords: crystallins • cataract • protein structure/function