Zinc finger constitutes one of the most common DNA binding motifs. Although zinc finger proteins consisting of Cys2His2, Cys3His, Cys4, and Cys6 domains are known in nature, a novel His4 zinc finger protein has never been observed. Herein, we have created the first artificial His4-type zinc finger protein (H4Sp1) engineered by Cys → His mutations of the Cys2His2-type zinc finger transcription factor Sp1. The CD features of the single finger H4Sp1f2 and three-finger H4Sp1 clearly demonstrate the folding of the mutant His4 peptides by complexation with Zn(II). The NMR study of Zn(II)-H4Sp1f2 reveals that some distortions of the helical region occur due to Zn(II) coordination. The gel mobility shift assay and DNase I footprinting analysis strongly show the binding of Zn(II)-H4Sp1 to the GC-box site of duplex DNA. The methylation interference pattern of Zn(II)-H4Sp1 binding significantly resembles that of the corresponding C2H2Sp1 binding. The present artificial peptide H4Sp1 is the first example of a zinc fi...