Chapter 4 │ Page 122 final equilibration, without enforcing any position restraints on the system, was performed for up to 350 ns, depending on the simulated system. Each system was simulated in triplicate, with di erent initial velocities, meaning a total of 12 systems (2 protein complexes, both in native and oxidized states) were used for our investigation. The final equilibration of each system was used to calculate the root-mean-square deviation (RMSD) of the alpha-carbons (Cα atoms) of the protein complexes. In addition, the last 100 ns were used to investigate the root-mean-square fluctuations (RMSF) of the protein residues, as well as the secondary structure of the fully equilibrated complexes and the salt bridge connections between ligand and receptor. For the latter, the VMD software [48] was used. 2.2. Investigating The Binding A inity Of The Complexes To investigate the binding a inity of the complexes, the fully equilibrated protein complex was placed in a new, triclinic simulation box that was elongated in the zdirection, and again solvated in SPC water containing 150 mM NaCl. The complex itself was oriented along the z-axis, i.e., the contact plane between the ligand and receptor was made approximately perpendicular to the z-axis. Next, a new energy minimization, NVT equilibration (2 ns) and NPT equilibration (30 ns) were performed, all while applying position restraints of 1000 kJ·mol-1·nm-2 to the heavy atoms of the protein. The complex was then pulled apart by subjecting the NK cell receptor (i.e., NKG2A/CD94 or KIR2DL1) to a harmonic potential with a force constant of 1000 kJ·mol-1·nm-2, at a constant velocity of 0.1 nm/ns for 40 ns. The center of mass of NKG2A/CD94 was pulled against the backbone atoms of Lys6, Tyr7, Phe8 and His9 of the HLA-E protein, while the center of mass of KIR2DL1 was pulled against the backbone atoms of Gln96, Arg97, Met98 and Phe99 of the HLA-Cw4. The above residues were chosen for each complex because they are buried inside the protein
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