FAST PHOTOCHEMICAL OXIDATION OF PROTEINS COUPLED WITH MASS SPECTROMETRY REVEALS CONFORMATIONAL STATES OF APURINIC/APYRIMIDIC ENDONUCLEASE 1

Abstract

Fast photochemical oxidation of proteins (FPOP) is an emerging footprinting method that utilizes hydroxyl radicals. The use of hydroxyl radicals create stable labeled products that can be analyzed with mass spectrometry. The advantage of FPOP over other methods is the fast acquisition of results and the small amount of sample required for analysis. Protein structure and protein- ligand interactions have been studied with FPOP. Here we evaluated (1) the reproducibility of FPOP, (2) the effect of hydrogen peroxide concentration on oxidation and (3) the use of FPOP to evaluate protein- nucleic acid interaction with Apurinic/Apurinic endonuclease 1 (APE1) protein. APE1 is a pleotropic protein that has been crystallized and studied widely. The 35641.5 Da protein has two major functional activities: DNA repair and redox function. An intact protein study of APE1 showed consistent global labeling by FPOP and a correlation between oxidation and hydrogen peroxide concentration. Furthermore, analysis of APE1 with DNA was done in hopes of probing the DNA binding site. Although the oxidation observed was not sufficient to define the complex pocket, a dramatic effect was seen in residue oxidation when DNA was added. Interestingly, the internal residues were labeled collectively in all APE1 experiments which indicates partial unfolding of the protein as previously suggested in the literature. Hence, these findings establish the use of FPOP to capture protein dynamics and provide evidence of the existence breathing dynamics of APE1.