RESUMO
Bimolecular fluorescence complementation (BiFC) assay has been used widely to visualize protein-protein interactions in cells. However, there is a problem that fluorescent protein fragments have an ability to associate with each other independent of an interaction between proteins fused to the fragments. To facilitate the BiFC assay, we have attempted to determine the structure and characteristics of reassembled fluorescent protein, Venus. The anion-exchange chromatography showed an oligomer and a monomer of reassembled Venus. Our results suggested that the oligomer was formed by ß-strands swapping without any serious steric clashes and was converted to the monomer. Crystal structure of reassembled Venus had an 11-stranded ß-barrel fold, typical of GFP-derived fluorescent proteins. Based on the structural features, we have mutated to ß-strand 7 and measured T(m) values. The results have revealed that the mutation influences the thermal stability of reassembled fluorescent complex.
