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  • Monitoring conformational changes in peroxisome proliferator-activated receptor α by a genetically encoded photoamino acid, cross-linking, and mass spectrometry.

Monitoring conformational changes in peroxisome proliferator-activated receptor α by a genetically encoded photoamino acid, cross-linking, and mass spectrometry.

Journal of medicinal chemistry (2013-05-04)
Rico Schwarz, Dirk Tänzler, Christian H Ihling, Mathias Q Müller, Knut Kölbel, Andrea Sinz
ABSTRACT

Chemical cross-linking combined with an enzymatic digestion and mass spectrometric analysis of the reaction products has evolved into an alternative strategy to structurally resolve protein complexes. We investigated conformational changes in peroxisome proliferator-activated receptor α (PPARα) upon ligand binding. Using E. coli cells with a special tRNA/aminoacyl-tRNA synthetase pair, two PPARα variants were prepared in which Leu-258 or Phe-273 were site-specifically replaced by the genetically encoded photoreactive amino acid p-benzoylphenylalanine (Bpa). PPARα variants were subjected to UV-induced cross-linking, both in the absence and in the presence of ligands. After the photo-cross-linking reaction, reaction mixtures were enzymatically digested and peptides were analyzed by mass spectrometry. The inter-residue distances disclosed by the photochemical cross-links served to monitor conformational changes in PPARα upon agonist and antagonist binding. The data obtained with our strategy emphasize the potential of genetically encoded internal photo-cross-linkers in combination with mass spectrometry as an alternative method to monitor in-solution 3D-protein structures.

MATERIALS
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Product Description

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