Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: Role of Arg 178 and Arg218 at the dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integ rity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactoba cillus casei was investigated by thermal and chemical denaturation. While t he R178F mutant showed apparent stability to thermal denaturation by its de creased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues re sults in more labile quaternary and tertiary interactions. Circular dichroi sm studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emph asize that quaternary interactions may influence the stability of the terti ary fold of TS, The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggre gated state of partially unfolded intermediate in the R178F mutant is stabl e over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 Angstrom crystal structure of the R 178F mutant reveals no major structural change as a consequence of amino ac id substitution. The results may be rationalized in terms of mutational eff ects on both the folded and unfolded state of the protein. Site specific am ino acid substitutions are useful in identifying specific regions of TS inv olved in association of non-native protein structures. Proteins 1999;34:356 -368. (C) 1999 Wiley-Liss, Inc.