Total synthesis of artificial zinc-finger proteins: Problems and perspectives.
The total synthesis of a peptide segment corresponding to the DNA-binding segment of Sp1 (positions 532-623) using a native chemical ligation approach is described. The folding of the synthetic segment in the presence of Zn(II) gave a zinc-coordinated protein. The dissociation constant (K(D)) for the DNA binding of the resulting protein, determined by a gel mobility shift assay, was 130 nM, almost nine times higher than that of the genetically prepared protein. However, methylation interference assay showed an identical sequence specificity of both proteins in DNA recognition. The chemical ligation method to connect the respective zinc-finger units was also accomplished. Successive ligation between a cysteine-containing peptide segment and a chloroacetylated peptide segment gave an artificial three-finger protein, which corresponds to the above DNA-binding segment of Sp1. However, this protein failed to bind DNA, even at 1.25 mM. Assessment of their folding structure based on the absorption spectra of their Co(II) complexes showed that the linker design to connect the respective finger units is critical for the proper folding of the proteins as well as the occurrence of the DNA-binding function.
Futaki
Shiroh
S
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Tatsuto
Kiwada
K
Shiraishi
Yasuhisa
Y
Sugiura
Yukio
Y
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000235:genetics; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D016329:Sp1 Transcription Factor; D004247:DNA; D003545:Cysteine