Effect of a single point mutation on the stability, residual structure and dynamics in the denatured state of GED: relevance to self-assembly

Abstract

The GTPase effector domain (GED) of dynamin forms large soluble oligomers in vitro, while its mutant - I697A - lacks this property at low concentrations. With a view to understand the intrinsic structural characteristics of the polypeptide chain, the global unfolding characteristics of GED wild type (WT) and I697A were compared using biophysical techniques. Quantitative analysis of the CD and fluorescence denaturation profiles revealed that unfolding occurred by a two-state process and the mutant was less stable than the WT. Even in the denatured state, the mutation caused chemical shift perturbations and significant differences were observed in the ¹⁵N transverse relaxation rates (R₂), not only at the mutation site but all around. These results demonstrate that the hydrophobic change associated with the mutation perturbs the structural and motional preferences locally, which are then relayed via different folding pathways along the chain and the property of oligomerization in the native state is affected.