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.