A tetrahedral zinc(II)-binding site introduced into a designed protein.
The ultimate goal of protein engineering is to create novel proteins which will adopt predetermined structures, bind specified ligands, and catalyze new reactions. Here we describe the successful introduction of metal-binding activity into a model four helix bundle protein. The designed binding site is tetrahedral and is formed by two Cys and two His ligands on adjacent helices. We have introduced this site into the protein and characterized the binding activity. Using 65Zn(II), we have shown that the protein binds Zn(II), that the sulfhydryls are essential for binding, and that binding occurs to the protein monomer. The designed protein binds metals with high affinity: we estimate the dissociation constants as 2.5 X 10(-8) M for Zn(II) and 1.6 X 10(-5) M for Co(II). The characteristic absorption spectrum of the Co(II)-substituted protein fully supports the model of a tetrahedral binding site comprised of two Cys and two His ligands. Circular dichroism studies indicate that no significant changes in secondary structure occur between the metal-bound and metal-free forms of the protein. However, the metal-bound form is substantially stabilized toward denaturation by GuHCl compared to the metal-free form.
Regan
L
L
Laboratory of Molecular Biology, Medical Research Council, Cambridge, U.K.
Clarke
N D
ND
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002413:Cations, Divalent; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003035:Cobalt; D003545:Cysteine; D006639:Histidine; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism
D002352:Carrier Proteins; D002413:Cations, Divalent; D008667:Metalloproteins; D011994:Recombinant Proteins; C098390:protein Z alpha 4; D003035:Cobalt; D006639:Histidine; D015032:Zinc; D003545:Cysteine