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P. Santhoshkumar, M. Raju, K. K. Sharma; Identification of Residues Involved in B-Crystallin Oligomerization - A Cysteine Scanning Mutagenesis Study. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1638.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have shown that several regions in B-crystallin are involved in subunit interaction. The objective of this study was to identify residues present in the subunit interface of B-crystallin using cysteine scanning mutagenesis and thiol cross-linking.
Single Cys mutations were introduced in human B-crystallin at I5, S19, L23, S35, T42, Y48, S59, M68, S76, K90, I98, S115, A126, T134, T144, S153, T162 and A171 positions using site-directed mutagenesis. The wild-type and mutant proteins were expressed in E coli BL21(DE3)pLysS cells and purified by gel filtration and ion-exchange chromatography. The wild-type and mutant proteins were characterized by spectroscopic methods and the chaperone activities of recombinant proteins were compared using alcohol dehydrogenase as client protein. The oligomeric masses of the proteins were estimated by multi-angle light scattering. SDS-PAGE analysis was performed under non-reducing conditions to evaluate the dimer formation propensity by various mutants. The extent of dimer formation was determined from the SDS-PAGE gels using KODAK image analysis software.
Far and near UV CD studies showed no major difference in the spectra of wild-type and mutant proteins. The average oligomeric mass of mutants under reducing condition was comparable to the oligomeric mass of B-crystallin. Except for cysteine mutants of the chaperone site (K90C and I98C) all other mutants spontaneously formed dimers with residues from the corresponding region of another subunit at room temperature. We found that the extent of dimer formation decreased when the mutation was introduced closer to the chaperone site. Subunits with Cys in 19-59 region exhibited strongest interaction (45-75% dimerization) whereas the Cys introductions in the C-terminal resulted in 5-25% dimers with the exception of T162C which showed 65% dimer formation.
Residues I5, S19, L23, S35, T42, Y48, S59, M68, S76, S115, A126, T134, T144, S153, T162 and A171 are located at the subunit interfaces in B-crystallin oligomers. The subunit interacting sites and the putative chaperone site are quite distinct. Our study shows that there is an ordered interaction of B-crystallin subunits during oligomerization.
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