Cu(II)-binding properties of a cytochrome c with a synthetic metal-binding site: His-X3-His in an alpha-helix.
A metal-binding site consisting of two histidines positioned His-X3-His in an alpha-helix has been engineered into the surface of Saccharomyces cerevisiae iso-1-cytochrome c. The synthetic metal-binding cytochrome c retains its biological activity in vivo. Its ability to bind chelated Cu(II) has been characterized by partitioning in aqueous two-phase polymer systems containing a polymer-metal complex, Cu(II)IDA-PEG, and by metal-affinity chromatography. The stability constant for the complex formed between Cu(II)IDA-PEG and the cytochrome c His-X3-His site is 5.3 x 10(4) M-1, which corresponds to a chelate effect that contributes 1.5 kcal mol-1 to the binding energy. Incorporation of the His-X3-His site yields a synthetic metal-binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility.
Todd
R J
RJ
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91126.
Van Dam
M E
ME
Casimiro
D
D
Haymore
B L
BL
Arnold
F H
FH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003300:Copper / Q000737:chemistry*; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D045304:Cytochromes c; D004795:Enzyme Stability; D014644:Genetic Variation; D006639:Histidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D003300:Copper; D045304:Cytochromes c