Variants of ribonuclease inhibitor that resist oxidation.
Human ribonuclease inhibitor (hRI) is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonucleases. hRI has 32 cysteine residues. The oxidation of these cysteine residues to form disulfide bonds is a rapid, cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence: Cys94 and Cys95, and Cys328 and Cys329. A cystine formed from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight-membered ring, which would disrupt the structure of hRI and could facilitate further oxidation. We find that replacing Cys328 and Cys329 with alanine residues has little effect on the affinity of hRI for bovine pancreatic ribonuclease A (RNase A), but increases its resistance to oxidation by 10- to 15-fold. Similar effects are observed for the single variants, C328A hRI and C329A hRI, suggesting that oxidation resistance arises from the inability to form a Cys328-Cys329 disulfide bond. Replacing Cys94 and Cys95 with alanine residues increases oxidation resistance to a lesser extent, and decreases the affinity of hRI for RNase A. The C328A, C329A, and C328A/C329A variants are likely to be more useful than wild-type hRI for inhibiting pancreatic-type ribonucleases in vitro and in vivo. We conclude that replacing adjacent cysteine residues can confer oxidation resistance in a protein.
Department of Biochemistry and Chemistry, University of Wisconsin-Madison, 53706-1544, USA.
B M
BM
Kim
L W
LW
Schultz
R T
RT
Raines
D000409:Alanine / Q000032:analysis; D003545:Cysteine / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008958:Models, Molecular; D010100:Oxygen / Q000378:metabolism; D017433:Protein Structure, Secondary; D012259:Ribonuclease, Pancreatic / Q000032:analysis*; D012260:Ribonucleases / Q000037:antagonists & inhibitors*
D006861:Hydrogen Peroxide; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D003545:Cysteine; D000409:Alanine; D010100:Oxygen