Development of ?-helical calpain probes by mimicking a natural protein-protein interaction.

ABSTRACT: We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an ?-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the ?-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing ?-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn ?-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs will be useful for future investigation of calpains, while the cross-linking technique will enable exploration of other protein-protein interactions.

SUBMITTER: Jo H 

PROVIDER: S-EPMC3523126 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature