title: Use of hybridization for distance measurement by fluorescence energy transfer in oligomeric proteins: distance between two functional sites in aspartase.
abstract: Our previous studies suggested that in a tetrameric enzyme aspartase, Cys-140 and Trp-430 are located at or near the catalytic and activator sites, respectively. To estimate the distance between these two sites, fluorescence energy transfer between a single tryptophan (Trp-430) and a fluorescent group specifically attached to Cys-140 has been measured. From the fluorescence spectra of the enzyme, the distance was calculated to be 22.2 A according to the Förster's theory. To estimate the contribution of energy transfer between subunits, we prepared hybrids composed of non-fluorescent and fluorescent subunits and showed that the energy transfer occurred mainly within one subunit in the tetramer. These results indicate that the use of hybridization is very effective as a general method for evaluation of intersubunit energy transfer in oligomeric proteins.
authors.affiliation: Department of Chemistry, Faculty of Science, Kyoto University, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Murase
authors.forename: Y
authors.initials: Y
authors.lastname: Kawata
authors.forename: N
authors.initials: N
authors.lastname: Yumoto
mesh_terms: D001220:Aspartate Ammonia-Lyase / Q000737:chemistry*; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004735:Energy Transfer; D005456:Fluorescent Dyes; D008301:Maleimides; D008956:Models, Chemical; D009154:Mutation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents; D014364:Tryptophan / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D008301:Maleimides; D013439:Sulfhydryl Reagents; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D014364:Tryptophan; D001220:Aspartate Ammonia-Lyase; D003545:Cysteine