| Autor: |
Chen XR; Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061, United States., Mercedes-Camacho AY; Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061, United States., Wilson KA; Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States., Bouchard JJ; Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States., Peng JW; Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States., Etzkorn FA; Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061, United States. |
| Abstrakt: |
Cell cycle regulatory enzyme Pin1 both catalyzes pSer/Thr- cis/trans -Pro isomerization and binds the same motif separately in its WW domain. To better understand the function of Pin1, a way to separate these activities is needed. An unnatural peptide library, R 1 CO-pSer-Pro-NHR 2 , was designed to identify ligands specific for the Pin1 WW domain. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite functional group was synthesized in one step from Wang resin. The SPPR was used in the preparation of the library by parallel synthesis. The final 315-member library was screened with our WW-domain-specific, enzyme-linked enzyme-binding assay (ELEBA). Four of the best hits were resynthesized, and the competitive dissociation constants were measured by ELEBA. NMR chemical-shift perturbations (CSP) of ligands with 15 N-labeled Pin1 were used to measure K d for the best four ligands directly, demonstrating that they were specific Pin1 WW domain ligands. Models of the ligands bound to the Pin1 WW domain were used to visualize the mode of binding in the WW domain. |
