Discussion
The denatured state D0 of a protein under native solvent conditions is important to determine its behavior in the cell, but it is usually hard to characterize because of its intrinsic instability and low population. By studying the dependence of NMR observables, like secondary chemical shifts and relaxation parameters, under different denaturing conditions and extrapolating their values to native conditions, we could provide a conformational characterization ofD0 of the HIV-1-PR1-95.
A remarkable result was that the extrapolations of these quantities to native conditions were rather independent on the denaturant and matched the minor population of D0 present at native conditions.
In 1976, Pfeil and Privalov showed in a series of experiments9 that the unfolding enthalpy of lysozyme, denatured by pH, GdmCl and temperature was identical, once the mean energy associated with the denaturant (e.g., the ionization energy in the case of pH) was subtracted. From this, they concluded that the states denatured by different means are thermodynamically indistinguishable. Ever since it has been discussed whether the denatured states generated by different means of denaturation were structurally different or not. In the present structural study, the extrapolation of chemical shifts to non-denaturing conditions plays a similar role to that of subtracting the denaturing energy in Privalov’s experiment and all the extrapolations seem to agree very well with the presence of a single denatured state.
The interpretation of the raw data produced by NMR experiments in terms of conformational properties of D0 is particularly difficult for a state composed of a plethora of heterogeneous conformations. In this case, MD simulations can be a valuable complement to the experimental data because of their ability to probe the system at atomic scale. A critical issue in this respect is whether MD simulations can provide a realistic picture of the state of interest of the protein. To address this concern, we compared the secondary chemical shifts, the hydrodynamic radius and the relaxation parameters predicted by the simulation with the experimental values. The good agreement we found is a consequence of two factors. First, we used advanced sampling techniques of simulation that favor the diffusion in the conformational space of the system, allowing it to sample an heterogeneous conformational space. Second, we employed a force field54 that was particularly adjusted to simulate intrinsically-disordered proteins54, namely systems with conformational properties that are analogous to those of the denatured state of a structured protein. It is important to stress that the tools to analyze a simulation of the denatured state are different than those typically used for native-like states. For example, while the commonly used RMSD is a poor quantifier of the similarity between pairs of conformations with subtle common features, the fraction q of common contacts being a more sensitive tool.
The detection of transient native and non-native structures in the denatured state of proteins is important to understand their fast folding65,66 and their aggregation4,67. In the case of a viral protein as HIV-1-PR, such structures can be also relevant as targets of antiviral molecules19–21. We found in D0specific secondary structures, both native and non-native, displaying an equilibrium probability of up to ≈30% and also specific tertiary structures with equilibrium probability of up to ≈10%. Among them, the most stable elements seem to be the native C-terminal α-helix and a non-native β-like structure at the center of the protein. In particular, our observations transient population of the hairpin β1-β2, the hairpin β4-β5, the hairpin β5-β6 and the terminal α-helix. Except for the C-terminal helix, the remaining structures correlate with the arches described by elevated R2 values (Fig. 4) and further suggest that the simulations are capturing the details of the ensemble. Furthermore, several substates of D0were identified by cluster analysis, each with peculiar conformational features, both native and non-native. Interestingly, we observed positive secondary chemical shift values for Cα and C at few places along the chain (Fig. 4), suggesting the presence of transient helicity in regions that in the native structure form β-strands. Thus, non-native interactions appear relevant to the unfolded state of HIV-1-PR and may play roles in guidance through the folding process.