Identification of interaction sites between human βA3- and αA/αB-crystallins by mammalian two-hybrid and fluorescence resonance energy transfer acceptor photobleaching methods

Abstract

Our recent study has shown that βA3-crystallin along with βB1-and β2-crystallins were part of high molecular weight complex obtained from young, old, and cataractous lenses suggesting potential interactions between α- and α-crystallins (Srivastava, O. P., Srivastava, K., and Chaves, J. M. (2008) Mol. Vis. 14, 1872-1885). To investigate this further, this study was carried out to determine the interaction sites of βA3-crystallin with αA- and βB-crystallins. The study employed a mammalian two-hybrid method, an in vivo assay to determine the regions of βA3-crystallin that interact with αA- and βB-crystallins. Five regional truncated mutants of βA3-crystallin were generated using specific primers with deletions of N-terminal extension (NT) (named βA3-NT), N-terminal extension plus motif I (named βA3-NT + I), N-terminal extension plus motifs I and II (named βA3-NT + I + II), motif III plus IV (named βA3-III + IV), and motif IV (named βA3-IV). The mammalian two-hybrid studies were complemented with fluorescence resonance energy transfer acceptor photobleaching studies using the above described mutant proteins, fused with DsRed (Red) and AcGFP fluorescent proteins. The results showed that the motifs III and IV of βA3-crystallin were interactive with αA-crystallin, and motifs II and III of βA3-crystallin primarily interacted with αB-crystallin. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.