Introduction
The denatured state D0 that proteins populate
transiently under native conditions1 is important to
determine their folding 2, stability3, aggregation4 and misfolding5, properties that can have direct implication for
disease states. Except for a few specific
proteins6–8, D0 is so poorly
populated that it escapes experimental observation. To overcome this
problem, induced denatured states can be stabilized by chemical agents
like urea, guanidine hydrochloride (GdmCl) or acids, populating the
states Durea , DGdmCl andDacid , respectively; states that are not
necessarily similar to D0 and which show
variation amongst themselves. However, from a thermodynamic point of
view, calorimetry experiments9 showed that the
unfolding enthalpy of lysozyme, denatured by pH, GdmCl and temperature
is identical once the energy associated with the denaturant mean (e.g.,
the ionization energy in the case of pH) was subtracted. From these
data, it was concluded that the states denatured by different means are
thermodynamically indistinguishable9.
One could then ask whether the conformational properties of the
different denatured states Durea ,DGdmCl , Dacid andD0 are similar as well. Although these states
were originally believed to be randomly disordered10,
recent studies have revealed them to contain transient
secondary11–15 and even tertiary
structures16,17. Such results were made possible
mainly thanks to the development of NMR techniques and in particular of
secondary chemical shift analysis.
In the present work, we studied the denatured states of a monomeric
variant of human immunodeficiency virus (HIV)-1 protease11This
is exactly the same protein used in ref. 27, in spite of the
unfortunate notation used in that reference.
(mHIV-1-PR1-95), a protein necessary for HIV-1 to
replicate in infected cells18. The denatured state of
HIV-1 protease under native conditions is particularly important because
it was suggested as a possible target of antiretroviral drugs that
prevent the correct folding of the protein and thus of its enzymatic
activity19–21. Moreover, the native conformation of
mHIV-1-PR1-95 displays a topology, which is more complex
than that of typical proteins of comparable size, a feature possibly
encoded also in its denatured state. In fact, its native conformation
displays two pseudo-knots and the associate Plaxco’s contact
order22, quantifying the non-locality of native
contacts, is 15, much larger than the values 8-10 of typical proteins of
comparable length.
HIV-1 protease is an aspartic acid protease, which in its active form
exists as a homodimer23 (Fig 1a). Analysis of its
folding kinetics identified a monomeric intermediate that associates to
form the native dimer structure24. Deletion of the
last four C-terminal residues stabilizes a monomeric, folded
form25. The native structure of this
mHIV-1-PR1-95, predominantly contains β-sheet structure
and a C-terminal α-helix18, highly similar to the
structure in the dimer (cf. Fig 1b). Both the unfolding and refolding
kinetics of mHIV-1-PR studied in urea by fluorescence display two time
scales, suggesting the presence of at least one kinetic intermediate and
the typical refolding time of mHIV-1-PR1-95 is of the
order of a minute24. Also, mechanical unfolding
experiments suggest the presence of folding and unfolding
intermediates26. Interestingly, mHIV-1-PR was shown to
display cold denaturation well above zero degrees
Celsius27, a feature that allowed us to compare the
denatured states Durea ,DGdmCl and Dacid to a
further state Dcold .
The native and non-native states of the wild-type and of several
variants of HIV-1-PR were also characterized both in silico andin vivo 28–30. In spite of its central role as
a target for anti-retroviral therapies, biochemical and biophysical data
on HIV-1 protease are still limited. A tethered dimer in
GdmCl31,32, a wild-type dimer in acetic
acid33 and HIV-1-protease embedded in its viral
precursor protein in urea 34 constitute some of these
states. However, none of these studies were performed on the same
variant of the protein, prohibiting a direct comparison of the results.