Figure 1: The native conformation of HIV-1-PR and mHIV-1-PR1-95. a) Structure of the HIV-1-PR homodimer (PDB code 1BVG). The active site is highlighted by yellow spheres. b) Structure of the 1-95 variant mHIV-1-PR1-95 (PDB code 1Q9P). c) The sequence of mHIV-1-PR; the active site is highlighted in yellow.
In the present work we have performed titration experiments in different chemical denaturants using the exact same mHIV-1-PR1-95variant. This allowed us to monitor how the conformational properties of the denatured state depend on the kind and concentration of the denaturing agent, eventually extrapolating the properties ofD0 . The main quantity we investigated was the secondary chemical shifts, measured by heteronuclear NMR experiments. A non-trivial problem one has to face is then to interpret these data in terms of conformational properties of the protein. To assist us in this goal, we performed advanced molecular dynamics simulations of mHIV-1-PR1-95 in water, building an ensemble of conformations that describes the denatured stateD0 . The correctness of the simulatedD0 was validated by back-calculating the secondary chemical shifts from the simulation and comparing them with those obtained from the extrapolation to zero denaturant of the NMR results.