title: Monitoring conformational changes in peroxisome proliferator-activated receptor α by a genetically encoded photoamino acid, cross-linking, and mass spectrometry.
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.
authors: Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, D-06120 Halle/Saale, Germany.
authors: Rico
authors: R
authors: Schwarz
authors: Dirk
authors: D
authors: Tänzler
authors: Christian H
authors: CH
authors: Ihling
authors: Mathias Q
authors: MQ
authors: Müller
authors: Knut
authors: K
authors: Kölbel
authors: Andrea
authors: A
authors: Sinz
mesh_terms: D001577:Benzophenones / Q000737:chemistry* / Q000528:radiation effects; D001665:Binding Sites; D002087:Butyrates / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D014644:Genetic Variation; D008024:Ligands; D008958:Models, Molecular; D010080:Oxazoles / Q000737:chemistry; D047493:PPAR alpha / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D010671:Phenylurea Compounds / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: C488060:4-benzoylphenylalanine; D001577:Benzophenones; D002087:Butyrates; D003432:Cross-Linking Reagents; C449302:GW 6471; C453899:GW 7647; D008024:Ligands; D010080:Oxazoles; D047493:PPAR alpha; D010671:Phenylurea Compounds; D014443:Tyrosine; D010649:Phenylalanine