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 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 pK(i). SPR measurements revealed that T has a high affinity for membranes, isolated or within the fusion proteins (K(D)< 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.
Chenal
Alexandre
A
Département d'Ingénierie et d'Etudes des Protéines, CEA-Saclay, 91191 Gif sur Yvette cedex, France.
Nizard
Philippe
P
Forge
Vincent
V
Pugnière
Martine
M
Roy
Marie-Odile
MO
Mani
Jean-Claude
JC
Guillain
Florent
F
Gillet
Daniel
D
D002942:Circular Dichroism; D004167:Diphtheria Toxin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D004167:Diphtheria Toxin; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins