Protein stabilization by engineered metal chelation.
A ligand can shift a protein's folding/unfolding equilibrium by binding with higher affinity to the native state. A metal-chelating site consisting of two histidines separated by three residues (His-X3-His) engineered into an alpha-helix provides a general and easily-implemented means for protein stabilization by this mechanism. We have tested this approach with the iso-1-cytochrome c of Saccharomyces cerevisiae substituted with histidine at positions 4 and 8 in its N-terminal alpha-helix. One mM Cu(II) complexed to iminodiacetate stabilizes the cytochrome c variant by ca. 1 kcal/mol, as determined by guanidinium chloride-induced unfolding. The protein's folding/unfolding equilibrium is shifted by a free energy equal to that calculated from the metal ion's preferential binding to the native protein. Given the ubiquity of surface alpha-helices and the additional possibility of inserting di-histidine chelating sites into turns and beta-structures, we conclude that this is a useful method for protein stabilization.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
J T
JT
Kellis
R J
RJ
Todd
F H
FH
Arnold
D001665:Binding Sites; D002614:Chelating Agents; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D004355:Drug Stability; D006639:Histidine; D008670:Metals; D011487:Protein Conformation; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
C014059:CYC1 protein, S cerevisiae; D002614:Chelating Agents; D003574:Cytochrome c Group; D008670:Metals; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D045304:Cytochromes c