Chapter 4 │ Page 128 considered as statistically significant. Data in the experimental graphs is represented as mean ± SEM, with individual values shown as dots in the bar plots. 3. RESULTS 3.1. Oxidation A ects The Protein Complex Structure, But Not The Binding Free Energy To gain insight into how NTP-induced oxidation a ects the MHC-I complexes HLACw4 and HLA-E, we performed non-reactive MD simulations for both the native and oxidized version of both protein complexes. To construct the oxidized versions of the ligands, we chose to implement the oxidation products of only the amino acids that are known to be most susceptible to NTP-oxidation, i.e. Met, Cys, Trp, Tyr, and Phe, that additionally have a high solvent accessibility in the simulated proteins, meaning they can be reached by RONS dissolved in the surrounding liquid during NTP treatment. Table 2 indicates which amino acids were oxidized in our simulated systems. Notably, all cysteine residues in both HLA-Cw4 and HLA-E have a very low solvent accessibility, thus preventing oxidation of the disulfide bonds present in the protein complexes. Table 2: List of amino acids used to construct the oxidized forms of the ligands (i.e. HLACw4 and HLA-E). Figure 3a shows the calculated RMSD of the simulated protein complexes in both native and oxidized form. Protein oxidation has been reported to cause conformational changes and to result in higher protein flexibility [30], as evident from G H I I H
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