Effects of Zn(II) binding and apoprotein structural stability on the conformation change of designed antennafinger proteins.
Ligand-induced conformation change is a general strategy for controlling protein function. In this work, we demonstrate the relationships between ligand binding and conformational stability using a previously designed protein, Ant-F, which undergoes a conformation change upon Zn(II) binding. To investigate the effect of stabilization of the apo structure on the conformation change, we also created a novel protein, Ant-F-H1, into which mutations are introduced to increase its stability over that of Ant-F. The chemical denaturation experiments clarified that apo-Ant-F-H1 is more stable than apo-Ant-F (DeltaDeltaG = -1.28 kcal/mol) and that the stability of holo-Ant-F-H1 is almost the same as that of holo-Ant-F. The Zn(II) binding assay shows that the affinity of Zn(II) for Ant-F-H1 is weaker than that for Ant-F (DeltaDeltaG = 1.40 kcal/mol). A large part of the increased value of free energy in stability corresponds to the decreased value of free energy in Zn(II) binding, indicating that the stability of the apo structure directly affects the conformation change. The denaturation experiments also reveal that Zn(II) destabilizes the conformation of both proteins. From the thermodynamic linkage, Zn(II) is thought to bind to the unfolded state with high affinity. These results suggest that the binding of Zn(II) to the unfolded state is an important factor in the conformational change as well as the stability of the apo and holo structures.
Hori
Yuichiro
Y
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Sugiura
Yukio
Y
D000595:Amino Acid Sequence; D051943:Antennapedia Homeodomain Protein; D001059:Apoproteins / Q000737:chemistry*; D001665:Binding Sites; D002942:Circular Dichroism; D003035:Cobalt / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D018398:Homeodomain Proteins / Q000138:chemical synthesis*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D020033:Protein Isoforms / Q000138:chemical synthesis; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014157:Transcription Factors; D015032:Zinc / Q000737:chemistry*; D016335:Zinc Fingers
D051943:Antennapedia Homeodomain Protein; D001059:Apoproteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D014157:Transcription Factors; D003035:Cobalt; D015032:Zinc; D019791:Guanidine