Exploring leucine‐rich repeat proteins as molecular scaffolds: design of an oxidation‐resistant ribonuclease inhibitor

Abstract

Engineered proteins with specific binding activities have evolved as a powerful technology. Repeat proteins, which exhibit structurally conserved modular frameworks and extensive surface areas, are alluring molecular scaffolds; some have yielded molecules with the specificity and robustness of antibodies. Our long‐term goal is to design novel binding surfaces on the human ribonuclease inhibitor (RI), a horseshoe‐shaped leucine‐rich repeat (LRR) protein that binds to pancreatic‐type ribonucleases with extraordinary affinity (KD ? fM). Exploration of RI as a protein scaffold has been limited by its oxidation sensitivity.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 Monticello College Foundation Research Grant to E.F.R., and start‐up funds (Macalester College and Lawrence University) to K.A.D.