Protein Engineering, Vol. 15, No. 5, 383-391,
May 2002
© 2002 Oxford University Press
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
1 Département d'Ingénierie et d'Etudes des Protéines, CEA-Saclay, 91191 Gif sur Yvette cedex, 2 Biophysique Moléculaire et Cellulaire, UMR 5090, Département de Biologie Moléculaire et Structurale, CEA-Grenoble,17 rue des Martyrs, 38054 Grenoble cedex 9 and 3 CNRS-UMR 5094, Faculté de Pharmacie, 15 avenue C. Flahault, 34093 Montpellier cedex 5, France
We investigated whether the structural and functional behaviors of two unrelated protein domains were modified when fused. The IgG-binding protein ZZ derived from staphylococcal protein A was fused to the N- and/or C-terminus of the diphtheria toxin transmembrane domain (T). T undergoes a conformational change from a soluble native state at neutral pH to a molten globule-like state at acidic pH, leading to its interaction with membranes. We found that this molten globule state was not connected to the GdnHCl-induced unfolding pathway of T. The pH-induced transition of T, and also the unfolding of T and ZZ at neutral and acidic pH, were unchanged whether the domains were isolated or fused. The position of ZZ, however, influenced the solubility of T near its pKi. SPR measurements revealed that T has a high affinity for membranes, isolated or within the fusion proteins (KD< 10-11 M). This work shows that in the case of T and ZZ, the fusion of protein domains with different stabilities does not alter the structural changes involved in folding and function. This supports the use of T as a soluble membrane anchor.
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