Does fusion of domains from unrelated proteins affect their folding pathways and the structural changes involved in their function? A case study with the diphtheria toxin T domain

We investigated whether the structural and functional behaviorsof two unrelated protein domains were modified when fused. TheIgG-binding protein ZZ derived from staphylococcal protein Awas fused to the N- and/or C-terminus of the diphtheria toxintransmembrane domain (T). T undergoes a conformational changefrom a soluble native state at neutral pH to a molten globule-likestate at acidic pH, leading to its interaction with membranes.We found that this molten globule state was not connected tothe GdnHCl-induced unfolding pathway of T. The pH-induced transitionof T, and also the unfolding of T and ZZ at neutral and acidicpH, were unchanged whether the domains were isolated or fused.The position of ZZ, however, influenced the solubility of Tnear its pK_i. SPR measurements revealed that T has a high affinityfor membranes, isolated or within the fusion proteins (K_D<10^-11 M). This work shows that in the case of T and ZZ, thefusion of protein domains with different stabilities does notalter the structural changes involved in folding and function.This supports the use of T as a soluble membrane anchor.