title: Electrocatalytic monitoring of metal binding and mutation-induced conformational changes in p53 at picomole level.
abstract: We developed an innovative electrochemical method for monitoring conformational transitions in proteins using constant current chronopotentiometric stripping (CPS) with dithiothreitol-modified mercury electrodes. The method was applied to study the effect of oncogenic mutations on the DNA-binding domain of the tumor suppressor p53. The CPS responses of wild-type and mutant p53 showed excellent correlation with structural and stability data and provided additional insights into the differential dynamic behavior of the proteins. Further, we were able to monitor the loss of an essential zinc ion resulting from mutation (R175H) or metal chelation. We envisage that our CPS method can be applied to the analysis of virtually any protein as a sensor for conformational transitions or ligand binding to complement conventional techniques, but with the added benefit that only relatively small amounts of protein are needed and instant results are obtained. This work may lay the foundation for the wide application of electrochemistry in protein science, including proteomics and biomedicine.
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic. palecek@ibp.cz
authors.forename: Emil
authors.initials: E
authors.lastname: Paleček
authors.forename: Veronika
authors.initials: V
authors.lastname: Ostatná
authors.forename: Hana
authors.initials: H
authors.lastname: Černocká
authors.forename: Andreas C
authors.initials: AC
authors.lastname: Joerger
authors.forename: Alan R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D002384:Catalysis; D004229:Dithiothreitol / Q000737:chemistry; D006801:Humans; D009154:Mutation; D011199:Potentiometry / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics; D015032:Zinc / Q000737:chemistry*
chemical_list: D016159:Tumor Suppressor Protein p53; D015032:Zinc; D004229:Dithiothreitol