title: An analytical workflow for the molecular dissection of irreversibly modified fluorescent proteins.
abstract: Owing to their ability to be genetically expressed in live cells, fluorescent proteins have become indispensable markers in cellular and biochemical studies. These proteins can undergo a number of covalent chemical modifications that may affect their photophysical properties. Among other mechanisms, such covalent modifications may be induced by reactive oxygen species (ROS), as generated along a variety of biological pathways or through the action of ionizing radiations. In a previous report [1], we showed that the exposure of cyan fluorescent protein (ECFP) to amounts of (•)OH that mimic the conditions of intracellular oxidative bursts (associated with intense ROS production) leads to observable changes in its photophysical properties in the absence of any direct oxidation of the ECFP chromophore. In the present work, we analyzed the associated structural modifications of the protein in depth. Following the quantified production of (•)OH, we devised a complete analytical workflow based on chromatography and mass spectrometry that allowed us to fully characterize the oxidation events. While methionine, tyrosine, and phenylalanine were the only amino acids that were found to be oxidized, semi-quantitative assessment of their oxidation levels showed that the protein is preferentially oxidized at eight residue positions. To account for the preferred oxidation of a few, poorly accessible methionine residues, we propose a multi-step reaction pathway supported by data from pulsed radiolysis experiments. The described experimental workflow is widely generalizable to other fluorescent proteins, and opens the door to the identification of crucial covalent modifications that affect their photophysics.
authors.affiliation: Laboratoire de Chimie Physique, UMR CNRS 8000, Building 350, 91405, Orsay Cedex, France.
authors.forename: Vivien
authors.initials: V
authors.lastname: Berthelot
authors.forename: Vincent
authors.initials: V
authors.lastname: Steinmetz
authors.forename: Luis A
authors.initials: LA
authors.lastname: Alvarez
authors.forename: Chantal
authors.initials: C
authors.lastname: Houée-Levin
authors.forename: Fabienne
authors.initials: F
authors.lastname: Merola
authors.forename: Filippo
authors.initials: F
authors.lastname: Rusconi
authors.forename: Marie
authors.initials: M
authors.lastname: Erard
mesh_terms: D000595:Amino Acid Sequence; D056148:Chromatography, Reverse-Phase; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000737:chemistry*; D054730:Protein Interaction Domains and Motifs; D011675:Pulse Radiolysis; D017382:Reactive Oxygen Species / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D017382:Reactive Oxygen Species; C527701:enhanced cyan fluorescent protein; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D010649:Phenylalanine; D008715:Methionine