Role of αBI5 and αBT162 residues in subunit interaction during oligomerization of αB-crystallin

PURPOSE: To determine whether the residues in the NH(2)- and COOH-terminal extensions interact with one another during oligomerization of αB-crystallin. METHODS: Site-directed mutagenesis was used to mutate αBI5 and αBT162 residues to Cys. The recombinant I5C and T162C proteins were expressed in Escherichia coli cells and purified using chromatographic techniques. These proteins were analyzed by SDS–PAGE and mass spectrometry and characterized by multi-angle light scattering and circular dichroism (CD) spectroscopy methods. Fluorescence resonance energy transfer (FRET) assay was used to determine the interaction between the subunits. RESULTS: Dimer formation was observed in both αBI5C and αBT162C in storage at 4 °C. During air oxidation at room temperature, αBT162C formed dimers to a greater extent than αBI5C. The average molar masses, secondary structures, and chaperone-like activities of the reduced forms of I5C and T162C were comparable to that of wild type αB-crystallin. The oligomeric assembly of reduced forms of I5C and T162C appeared homogenous under JEOL 1200EX Electron microscope whereas the oxidized proteins appeared as irregular aggregates. FRET assay demonstrated interactions between αBI5C–αBI5C and αBT162C–αBT162C. However, there was no evidence of an interaction between αBI5C and αBT162C residues during oligomerization. CONCLUSIONS: This study suggests that residues from the NH(2)- and COOH-terminal regions in αB-crystallin interact with residues from the corresponding regions of another subunit, but there exists no interaction between the residues at the COOH-terminal extension region and the residues at the NH(2)-terminal region.