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Production and characterization of oxidation‐resistant variants of the human ribonuclease inhibitor
Author(s) -
Uebersohn Alec W,
Dickson Kimberly A.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.996.14
Subject(s) - protein engineering , amino acid , ribonuclease , chemistry , leucine rich repeat , cysteine , biochemistry , peptide , computational biology , biology , enzyme , rna , gene
Repeat proteins are widely used in nature to modulate protein activity. New protein engineering technologies have exploited the scaffolds of repeat proteins to construct novel protein‐binding surfaces that rival the specificity and robustness of antibodies. The human ribonuclease inhibitor (RI) is a horseshoe‐shaped leucine‐rich repeat (LRR) protein that binds to pancreatic‐type ribonucleases with extraordinary affinity ( K D ≈ fM). Its conserved modular frameworks and extensive surface area renders it an alluring scaffold for protein engineering. Unfortunately, RI is extremely sensitive to oxidation due to the high number of cysteines (32 residues, 7 % of total amino acids). Oxidation of only a few cysteines results in a collapse of the tertiary structure and complete inactivation. We have constructed oxidation‐resistant variants of RI in which some or all of its 32 cysteine residues have been replaced. Six variants of RI have been produced in which groups of 5–7 cysteines (at conserved positions within the LRR motif) have been mutated. Preliminary results indicate that the RI variants maintain affinity for ribonucleases, but possess decreased oxidation sensitivity. These data contribute to our understanding of the structural stability of LRR proteins and suggest that RI and other LRR proteins are practical scaffolds for the design of novel binding surfaces. This work was supported by a Lawrence University Excellence in Science Grant and a Mellon Foundation Grant for Senior Experience to A.W.U and Lawrence University start‐up funds to K.A.D.