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.