title: Structure and characteristics of reassembled fluorescent protein, a new insight into the reassembly mechanisms.
abstract: 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 β-strands swapping without any serious steric clashes and was converted to the monomer. Crystal structure of reassembled Venus had an 11-stranded β-barrel fold, typical of GFP-derived fluorescent proteins. Based on the structural features, we have mutated to β-strand 7 and measured T(m) values. The results have revealed that the mutation influences the thermal stability of reassembled fluorescent complex.
authors.affiliation: Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
authors.forename: Masami
authors.initials: M
authors.lastname: Isogai
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kawamoto
authors.forename: Kazuto
authors.initials: K
authors.lastname: Inahata
authors.forename: Harumi
authors.initials: H
authors.lastname: Fukada
authors.forename: Kenji
authors.initials: K
authors.lastname: Sugimoto
authors.forename: Toshiji
authors.initials: T
authors.lastname: Tada
mesh_terms: D018360:Crystallography, X-Ray; D005456:Fluorescent Dyes / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*; D008958:Models, Molecular
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins