Perfluoro-tert-Butyl Hydroxyprolines as Sensitive, Conformationally Responsive Molecular Probes: Detection of Protein Kinase Activity by 19F NMR.
Related Articles Perfluoro-tert-Butyl Hydroxyprolines as Sensitive, Conformationally Responsive Molecular Probes: Detection of Protein Kinase Activity by 19F NMR.
ACS Chem Biol. 2020 Mar 03;:
Authors: Tressler CM, Zondlo NJ
Abstract
19F NMR spectroscopy provides the ability to quan-titatively analyze single species in complex solu-tions, but is often limited by the modest sensitivity inherent to NMR. 4R- and 4S-Perfluoro-tert-buyl hydroxyproline contain 9 equivalent fluorines, in amino acids with strong conformational prefer-ences. In order to test the ability to use these amino acids as sensitive probes of protein modifications, the perfluoro-tert-buyl hydroxyprolines were in-corporated into substrate peptides of the protein kinases PKA and Akt. Peptides containing each dia-stereomeric proline were rapidly phosphorylated by each protein kinase and exhibited 19F chemical shift changes as a result of phosphorylation. The sensi-tivity of the perfluoro-tert-butyl group allowed quantitative analysis of the kinetics of phosphoryla-tion over three half-lives at single-digit micromolar concentrations of each species. The distinct con-formational preferences of these amino acids al-lowed the optimization of the substrate with a con-formationally matched amino acid, in order to maximize the rate of phosphorylation. PKA pre-ferred the 4R-amino acid at the -1 position, where-as the closely related AGC kinase Akt preferred the 4S-amino acid. These data, combined with analysis of structures of the Michaelis complexes of these kinases in the PDB, suggest that PKA recognizes the PPII conformation at the P-1 position relative to the phosphorylation site, while Akt/PKB recognizes an extended conformation at this position. These results suggest that conformational targeting may be employed to increase specificity in recognition by protein kinases. Perfluoro-tert-butyl hydroxypro-lines were applied to the real-time detection and quantification of PKA activity and inhibition of PKA activity in HeLa cell extracts via 19F NMR spectroscopy. The coupling of proline ring pucker with main chain conformation suggests broad ap-plication of perfluoro-tert-butyl hydroxyprolines in molecular sensing and imaging.
PMID: 32125821 [PubMed - as supplied by publisher]
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