Structural basis of protein oxidation resistance: a lysozyme study

Abstract

Accumulation of oxidative damage in proteins correlates with aging since it can cause irreversible and progressive degeneration of almost all cellular functions. Apparently, native protein structures have evolved intrinsic resistance to oxidation since perfectly folded proteins are, by large most robust. Here we explore the structural basis of protein resistance to radiation-induced oxidation using chicken egg white lysozyme in the native and misfolded form. We study the differential resistance to oxidative damage of six different parts of native and misfolded lysozyme by a targeted tandem/mass spectrometry approach of its tryptic fragments. The decay of the amount of each lysozyme fragment with increasing radiation dose is found to be a two steps process, characterized by a double exponential evolution of their amounts: the first one can be largely attributed to oxidation of specific amino acids, while the second one corresponds to further degradation of the protein. By correlating these results to the structural parameters computed from molecular dynamics (MD) simulations, we find the protein parts with increased root-mean-square deviation (RMSD) to be more susceptible to modifications. In addition, involvement of amino acid side-chains in hydrogen bonds has a protective effect against oxidation Increased exposure to solvent of individual amino acid side chains correlates with high susceptibility to oxidative and other modifications like side chain fragmentation. Generally, while none of the structural parameters alone can account for the fate of peptides during radiation, together they provide an insight into the relationship between protein structure and susceptibility to oxidation.

Figure 1. Workflow of the analytical method.

Figure 2. Analysis of the reduced-alkylated lysozyme irradiated with a dose at 1000 Gy.

(a) TIC chromatogram, (b) CID spectrum of the CELAAAMK ion m/z 447.2 detected between 8.8 and 9.9 min and (c) CID spectrum of the CELAAAMK ion m/z 455.2 detected between 6.8 and 7.2 min.

Figure 3. Evolution of the signal of the CELAAAMK peptide (a, b) and oxidized CELAAAMK peptide (c, d).

CELAAAMK peptide (3 transitions) irradiated in the native (a) and reduced-alkylated form (b) Oxidized CELAAAMK peptide (3 transitions) irradiated in the native (c) and reduced-alkylated form (d).

Figure 4. Lysozyme tertiary structure (PDB:1AKI).

(a) CELAAAMK is shown in yellow, GTDVQAWIR in green, HGLDNYR in white, GYSLGNWVCAAK in blue, FESNFNTQATNR in pink and WWCNDGR in red. (b) 180° degree rotation around y-axis.

Figure 5. Sequence of the chicken lysozyme with, highlighted in blue, the detected peptides.