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M. Raju, P. Santhoshkumar, K. K. Sharma; Role of I5 and T162 of Human B-crystallin in Subunit Interaction and Oligomerization. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2035.
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The purpose of the study was to investigate the role of N terminal and C terminal residues during αB crystallin oligomerization.
Substitution of I5 (N terminal) and T162 (C terminal) with cysteine was performed using site directed mutagenesis. The recombinant proteins were expressed in E.coli cells and purified using chromatographic techniques. These proteins were analyzed by SDS-PAGE and Mass Spectrometry and characterized by Dynamic Light Scattering and Circular dichroism (CD) spectroscopy methods. The oligomeric structure of the mutant αB-crystallin was observed by JEOL 1200EX Electron microscope. The interaction between I5C and T162C was analyzed by FRET assay and Electrospray mass spectrometry.
The average molar mass and the secondary structure and chaperone-like activity of I5C and T162C of the reduced forms were comparable to that of wild type αB-crystallin. Spontaneous dimer formation was observed in both I5C and T162C on storage at 4°C and accelerated dimer formation was seen with T162C compared to I5C subjected to air oxidation at room temperature. Under Electron Microscope the oligomeric assembly of reduced form of I5C and T162C appeared homogenous compare to oxidize forms, which showed aggregated species. The FRET assay confirmed the interaction between subunits at residues I5 and T162. However, there was no evidence of interaction between I5C and T162C.
The I5 and T162 are involved in subunit interaction, but there is no interaction between I5 and T162 during oligomeric assembly of αB crystallin protein.
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