The denatured state of HIV-1 protease under native conditions.

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.

Cold Denaturation of the HIV-1 Protease Monomer.

The human immunodeficiency virus-1 (HIV-1) protease is a complex protein that in its active form adopts a homodimer dominated by ß-sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1 protease that is populated above 0 °C and therefore directly accessible to various spectroscopic approaches. Using nuclear magnetic resonance secondary chemical shifts, temperature coefficients, and protein dynamics, we suggest that the cold-denatured state populates a compact wet globule containing transient non-native-like α-helical elements. From the linearity of the temperature coefficients and the hydrodynamic radii, we propose that the overall architecture of the cold-denatured state is maintained over the temperature range studied.

The maturation of HIV-1 protease precursor studied by discrete molecular dynamics.

The equilibrium properties of a HIV-1-protease precursor are studied by means of an efficient molecular dynamics scheme, which allows for the simulation of the folding of the protein monomers and their dimerization into an active form and compare them with those of the mature protein. The results of the model provide, with atomic detail, an overall account of several experimental findings, including the NMR conformation of the mature dimer, the calorimetric properties of the system, the effects of the precursor tail on the dimerization constant, the secondary chemical shifts of the monomer, and the paramagnetic relaxation enhancement data associated with the conformations of the precursor. It is found that although the mature protein can dimerize in a unique, single way, the precursor populates several dimeric conformations in which monomers are always native-like, but their binding can be non-native.

Identification of the folding inhibitors of hen-egg lysozyme: gathering the right tools.

The unfolded state of proteins displays a surprisingly rich amount of local native structure, which appears to be critical for driving the protein to its native state. Peptides with the same sequence of the corresponding structured segments can be used to interfere with the correct folding of the protein. Using model simulations, we investigate the folding of hen-egg lysozyme, identifying its key segments. Activity assays, NMR and circular dichroism experiments are used to screen the peptides which are able to inhibit the folding of lysozyme. Few peptides, corresponding to the segments of the protein which are structured in the unfolded state, are identified to have significant inhibitory effects.