Calcium ion responsive DNA binding in a zinc finger fusion protein.
Zinc finger fusion proteins, having a Ca-binding site from troponin C, were created to develop Ca-responsive regulation of DNA binding. The typical zinc finger folding of a novel fusion protein with a single finger, F2-Tn, was investigated using UV-vis spectroscopy of the Co-substituted form and CD experiments. Detailed structural analyses of F2-Tn/Zn2+ using NMR experiments and structural calculations clarify that our fusion protein gives a native zinc finger folding with the artificial Ca-binding domain intervening two helices. The Ca-responsive DNA-binding affinity of troponin-fused protein with two fingers (using F1F2-Tn) was investigated by electrophoretic mobility shift assay (EMSA). EMSA analyses of F1F2-Tn were performed under the conditions of various concentrations of the Ca ion. F1F2-Tn has a Kd value of 5.8 nM in the absence of Ca ion and shows a higher Kd value of 13 nM in the presence of 100 equiv of Ca ion. The artificially designed fusion zinc finger protein with a Ca-binding domain has Ca-responsive DNA-binding affinity. It is leading to a better understanding of the construction of zinc finger-based artificial transcriptional factors with a Ca switch.
Onoda
Akira
A
Department of Chemistry, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601, Japan.
Arai
Nozomi
N
Shimazu
Naoto
N
Yamamoto
Hitoshi
H
Yamamura
Takeshi
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012996:Solutions / Q000737:chemistry; D019209:Troponin C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics* / Q000502:physiology*
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012996:Solutions; D019209:Troponin C; D004247:DNA; D002118:Calcium