Chapter 4 │ Page 123 under the ligand-receptor binding domain, are approximately centered around the z-axis, and exhibit minimal fluctuations compared to other residues in the protein. To prevent movement of the ligand while retaining its flexibility, position restraints were applied to the Cα atoms of Phe36, Cys101, Phe116 and Cys203 (for HLA-E) and Glu63, Tyr118, Tyr159 and Ala205 (for HLA-Cw4). These residues were again chosen as they are buried inside the protein and exhibit low fluctuation. Finally, to prevent movement of the pulled protein (i.e. NKG2A/CD94 or KIR2DL1) in the xyplane, so-called flat-bottomed position restraints were applied with a radius of 0.05 nm and a force constant of 500 kJ·mol-1·nm-2. Along the reaction coordinate of each pulling simulation, 40 frames (later supplemented up to 53 frames to ensure adequate sampling) separated by 0.1 nm were isolated to serve as the initial structure in US simulations [49] to sample conformational space in a window at that pulled distance. For the native systems, US was performed for 50 ns, while for the oxidized complexes the US was performed for 75 ns. The last 25 ns of each US simulation were used for data collection (while the first part of the US was used to equilibrate the frame). To extract the free energy profiles along the pulling coordinate, the weighted histogram analysis method (WHAM) was employed [50]. 2.3. Building The Oxidized Structures The oxidized version of both investigated protein complexes was constructed with the Vienna-PTM web server [51] by replacing relevant native amino acids with their oxidized form. Which amino acids would be oxidized by NTP treatment was determined based on two factors: (i) the susceptibility of di erent amino acids to oxidation, and (ii) the accessibility of these amino acids in the investigated proteins to the solvent, and thus to plasma-produced RONS. Di erent amino acids have di erent susceptibility to PTM after plasma treatment. Takai et al. [52] experimentally investigated the chemical modification of amino
RkJQdWJsaXNoZXIy MTk4NDMw