Modifications generated by fast photochemical oxidation of proteins reflect the native conformations of proteins

Abstract

Hydroxyl radical footprinting (HRF) is a nonspecific protein footprinting method that has been increasingly used in recent years to analyze protein structure. The method oxidatively modifies solvent accessible sites in proteins, which changes upon alterations in the protein, such as ligand binding or a change in conformation. For HRF to provide accurate structural information, the method must probe the native structure of proteins. This requires careful experimental controls since an abundance of oxidative modifications can induce protein unfolding. Fast photochemical oxidation of proteins (FPOP) is a HRF method that generates hydroxyl radicals via photo-dissociation of hydrogen peroxide using an excimer laser. The addition of a radical scavenger to the FPOP reaction reduces the lifetime of the radical, limiting the levels of protein oxidation. A direct assay is needed to ensure FPOP is probing the native conformation of the protein. Here, we report using enzymatic activity as a direct assay to validate that FPOP is probing the native structure of proteins. By measuring the catalytic activity of lysozyme and invertase after FPOP modification, we demonstrate that FPOP does not induce protein unfolding.

Fast photochemical oxidation of proteins (FPOP): A powerful mass spectrometry–based structural proteomics tool

“…A radical scavenger, most commonly glutamine, is also added to the sample as another experimental control to prevent over-oxidation. Based on the reactivity of glutamine with OH, FPOP labels proteins on the microsecond timescale and ensures labeling of the native state of proteins (41,42).…”

Evolution of Structural Biology through the Lens of Mass Spectrometry

“…6 One HRF method, fast photochemical oxidation of proteins (FPOP) utilizes a pulsed laser, which is used to photolyze hydrogen peroxide on a microsecond time scale, which is faster than protein unfolding. 109,110 FPOP has been applied to many purified protein complexes and systems, revealing a wealth of information on protein structure and protein–protein interactions. 108,110–113…”

“…109,110 FPOP has been applied to many purified protein complexes and systems, revealing a wealth of information on protein structure and protein–protein interactions. 108,110–113…”

Protein profiling and pseudo-parallel reaction monitoring to monitor a fusion-associated conformational change in hemagglutinin

“…To overcome these challenges, oxidative and electrophilic labeling approaches have been taken to footprint water accessibility on protein surfaces, both in buffer and in complex media [6][7][8][9][10]. Among oxidative approaches, SPROX employs peroxide to oxidize methionine residues [9,11], while FPOP generates oxidizing radicals through photolysis [12][13][14][15]. Electrophilic approaches include targeting lysine with NHS esters [16,17], oxyimidates, and thioimidates [18][19][20], and targeting glutamate and aspartate with glycine ethyl ester [21][22][23].…”