A direct comparison of the properties of natural and designed zinc-finger proteins.
BACKGROUND
Zinc-finger proteins of the Cys2His2 type constitute an important family of DNA-binding proteins. Each zinc-finger domain has three residues that are thought to be important in determining DNA binding site specificity. Proteins have been designed previously by combining zinc-finger domains with a fixed sequence framework with different DNA-contacting residues.


RESULTS
We compared the DNA-binding properties of the DNA-binding domain from the human transcription factor Sp1, which contains three zinc fingers, with designed proteins in which the sequences of the structural framework were greatly modified but the presumed DNA-contacting residues were retained. Frameworks based on a zinc-finger consensus sequence and on a minimalist sequence consisting largely of alanine residues were studied. The preference for binding to the target sequence, 5'-(G,T)GG G(C,A)G GG(G,T)-3', was retained in all cases tested. The consensus framework-based protein was found to be superior to the natural one in terms of overall DNA-binding affinity, the degree of sequence discrimination, and the resistance to inactivation by chelating agents.


CONCLUSIONS
Our observations provide direct evidence that the residues previously observed to interact with the DNA bases are indeed the most important residues for determining DNA-binding specificity. We have also shown that these domains can tolerate considerable sequence variation while retaining function as well as three-dimensional structure. Finally, they show that framework modification can be used to generate proteins that have normal or enhanced DNA-binding activity but have different metal-binding properties.
Shi
Y
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Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Berg
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D013816:Thermodynamics; D016335:Zinc Fingers / Q000502:physiology*
D002614:Chelating Agents; D004268:DNA-Binding Proteins; D004247:DNA; D004492:Edetic Acid