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.