COVALENT TETHERING OF THE DIMER INTERFACE ANNULS AGGREGATION IN THYMIDYLATE SYNTHASE

Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggre gates at concentrations of urea (3.3-5 M), well below that required fo r complete denaturation, as established by fluorescence and size-exclu sion chromatography. In contrast to the wild-type enzyme, an engineere d mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are c rosslinked by disulfide bridges between residues 155-188' and 188-155' , does not show this behavior. Aggregation behavior is restored upon d isulfide bond reduction in the mutant protein, indicating the involvem ent of interface segments in forming soluble associated species. Inter molecular disulfide crosslinking has been used as a probe to investiga te the formation of larger non-native aggregates. The studies argue fo r the formation of large multimeric species via a sticky patch of poly peptide from the dimer interface region that becomes exposed on partia l unfolding. Covalent reinforcement of relatively fragile protein-prot ein interfaces may be a useful strategy in minimizing aggregation of n on-native structures in multimeric proteins.