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
V. Prasanna et al., 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, PROTEINS, 34(3), 1999, pp. 356-368
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.