Development of 伪-Helical Calpain Probes by Mimicking a Natural Protein鈥揚rotein Interaction

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• 作者：Hyunil Jo ; Nataline Meinhardt ; Yibing Wu ; Swapnil Kulkarni ; Xiaozhen Hu ; Kristin E. Low ; Peter L. Davies ; William F. DeGrado ; Doron C. Greenbaum
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：October 24, 2012
• 年：2012
• 卷：134
• 期：42
• 页码：17704-17713
• 全文大小：482K
• 年卷期：v.134,no.42(October 24, 2012)
• ISSN：1520-5126

文摘

We have designed a highly specific inhibitor of calpain by mimicking a natural protein鈥損rotein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an 伪-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 伪-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing 伪-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 伪-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鈥損rotein interactions.