Effects Of Reactive Oxygen Species On Cyan Fluorescent Protein

Abstract

Recent advances in microscopy techniques and the development of many different colour variants of the GFP family of proteins allow for a more direct analysis of protein function in live cells. The advantage of genetically coded fluorescent protein probes is often offset by their photophysical properties which usually make them very sensitive to cellular environmental changes.Among these, reactive oxygen species (ROS) are an essential part of key cellular processes (mitochondria respiration, apoptosis) and are also involved in the pathogenesis of various diseases (cancer, atherosclerosis, Alzheimer's disease, etc.). We studied the effects of ROS on the cyan fluorescent protein (CFP) in vitro, as this fluorescent protein is currently one of the most widely used in protein interaction studies. We studied the fluorescence and absorption changes of recombinant CFP protein using γ-radiolysis for ROS production. γ-radiolysis ROS production allowed us to have an exact control over the radical concentrations delivered unto the protein samples. The radicals used in this study were OH°, O2− or a mixture of OH° and O2−. We also determined the chemical modifications that take place upon ROS induced protein oxidation by mass spectrometry. We show that the targets of oxidation are one tyrosine and four methionine residues located on the protein surface and that the chromophore is not likely modified through these oxidation processes.