OLIGOMERIZATION OF THE CYTOPLASMIC FRAGMENT FROM THE ASPARTATE RECEPTOR OF ESCHERICHIA-COLI

We have observed that a 31-kDa cloned fragment from the Escherichia coli aspartate receptor exhibits a reversible monomer-oligomer reaction. The fragment, derived from the cytoplasmic region of the receptor (c-fragment), contains the signaling functions of the receptor. The wild-type and nine missense mutant fragments were analyzed. The latter were selected by the effect of the mutations on the signaling properties of the intact receptor, which induced either persistent smooth swimming or tumbling in bacteria [Mutoh, N., Oosawa, K., & Simon, M. I. (1986) J. Bacteriol. 167, 992-998]. In pH 7.0 buffer, the mutations caused five out of the six smooth mutant c-fragments to form oligomers, while neither the three tumble mutant nor wild-type fragments exhibited significant oligomer formation. At a lower pH (5.5), all of the fragments displayed some tendency to form oligomers. The equilibria between the monomer and the oligomers were monitored by gel permeation chromatography (GPC) which resolved two to three forms with apparent molecular weights between 110 000 and 270 000. The proportions of the different forms depended on concentration, indicating an association-dissociation reaction. Static light scattering (SLS) was used to demonstrate that the solution molecular mass of the wild-type c-fragment was 31 kDa and not 110 kDa as indicated by chromatography. One oligomer-forming c-fragment (S461L) eluted as the monomer and one other form, which was determined to be a dimer by SLS. The weight-average molecular weights, calculated from GPC data as a function of protein concentration, agreed well with the weight-average molecular weights obtained by SLS for this mutant. The GPC data indicate that the c-fragment forms oligomers of well-defined stoichiometry, and the anomalously large molecular weight estimates indicate that the fragment is extremely nonspherical in shape. The correlation between the behavioral phenotype and the tendency to form oligomers suggests that the mutations can indeed lock the receptor into well-defined signaling forms and that subunit interactions in the cytoplasmic region are stronger in the attractant-bound form of the receptor. The formation of the oligomers may have important implications for the mechanism of transmembrane signaling and is consistent with a mechanism that involves ligand-induced receptor clustering.