Abstract
The denatured state of several proteins has been shown to display transient structures that are relevant for folding, stability, and aggregation. To detect them by nuclear magnetic resonance (NMR) spectroscopy, the denatured state must be stabilized by chemical agents or changes in temperature. This makes the environment different from that experienced in biologically relevant processes. Using high‐resolution heteronuclear NMR spectroscopy, we have characterized several denatured states of a monomeric variant of HIV‐1 protease, which is natively structured in water, induced by different concentrations of urea, guanidinium chloride, and acetic acid. We have extrapolated the chemical shifts and the relaxation parameters to the denaturant‐free denatured state at native conditions, showing that they converge to the same values. Subsequently, we characterized the conformational properties of this biologically relevant denatured state under native conditions by advanced molecular dynamics simulations and validated the results by comparison to experimental data. We show that the denatured state of HIV‐1 protease under native conditions displays rich patterns of transient native and non‐native structures, which could be of relevance to its guidance through a complex folding process.
Highlights
The denatured state D0 that proteins populate transiently under native conditions[1] is important to determine their folding,[2] stability,[3] aggregation,[4] and misfolding;[5] properties that can have direct implication for disease states
Using high-resolution heteronuclear nuclear magnetic resonance (NMR) spectroscopy, we have characterized several denatured states of a monomeric variant of human immunodeficiency virus (HIV)-1 protease, which is natively structured in water, induced by different concentrations of urea, guanidinium chloride, and acetic acid
We show that the denatured state of HIV-1 protease under native conditions displays rich patterns of transient native and non-native structures, which could be of relevance to its guidance through a complex folding process
Summary
The denatured state D0 that proteins populate transiently under native conditions[1] is important to determine their folding,[2] stability,[3] aggregation,[4] and misfolding;[5] properties that can have direct implication for disease states. Analysis of its folding kinetics identified a monomeric intermediate that associates to form the native dimer structure.[24] Deletion of the last four C-terminal residues stabilizes a monomeric, fully folded form.[25] the native structure of this mHIV-1-PR1–95, which predominantly contains β-sheet structure and a C-terminal α-helix,[18] is highly similar to the structure in the dimer (cf Figure 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 minute.[24] mechanical unfolding experiments suggest the presence of folding and unfolding intermediates.[26] Interestingly, mHIV-1-PR was shown to display cold denaturation well above zero degrees Celsius,[27] a feature that allowed us to compare the denatured states Durea, DGdmCl, and Dacid to a further state Dcold. The correctness of the simulated D0 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
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