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.

Keywords: fast photochemical oxidation of protein (FPOP); hydroxyl radical footprinting (HRF); mass spectrometry; native protein structure; oxidative modifications; protein footprinting.

Figure 1

(A) Absorbance at 450 nm was tested every minute for 15 minutes to determine the activity of lysozyme. The highest activity was in the folded lysozyme sample followed by typcial FPOP control, quench condition, over‐oxidized control, typical FPOP sample, over‐oxidized sample, and unfolded sample, respectively. (B) For each condition, the activity was normallized using their protein concentration and compared to the folded lysozyme, which was set to 100% activity. (C) Extent of FPOP modifications for lysozyme on the residue‐level for the typical FPOP and over‐oxidization condition. Red boxes highlight the residues involved with catalytic activity and purple boxes highlight the buried residues.

Figure 2

Intact MS analysis show multiple oxidation states of lysozyme after FPOP in the (A) over‐oxidized sample and (B) typical FPOP sample.

Figure 3

FPOP modifications from lysozyme mapped on a crystal structure (pdb ID: 4WMG) for the (A) typical FPOP condition and (B) over‐oxidized condition. The modified active site residues are highlighted in red, and the modified residues with a SASA value ≤0.11 are highlighted in purple.

Figure 4

(A) Normalized catalytic activity of invertase with folded protein set to 100% activity. (B‐D) Extent of FPOP modifications for invertase on the residues level for typical FPOP condition and over‐oxidized condition.

Figure 5

FPOP modifications from invertase mapped on a crystal structure (pdb ID: 4EQV) for the (A) typical FPOP condition and (B) over‐oxidized condition. The modified residue in the active site is highlighted in red.

Figure 6

The MS/MS spectra for the (A) unmodified peptide and the (B) modified peptide. A 44 Dalton lose for both the b₅ and y₁₀ ion on the modified peptide corresponds to a CO₂ loss on Asp42.