A hypothesis to reconcile the physical and chemical unfolding of proteins

High pressure (HP) or urea is commonly used to disturb folding species. Pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the protein–solvent system. However, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein–urea interactions are considered to be crucial. Here, we provide NMR spectroscopy and 3D reconstructions from X-ray scattering to develop the “push-and-pull” hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by HP. In studying MpNep2 from Moniliophthora perniciosa, we tracked two cooperative units using HP-NMR as MpNep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. At subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by NMR intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle X-ray scattering (SAXS)]. This state has a higher susceptibility to pressure denaturation (lower p_1/2 and larger ΔV_u); thus, urea and HP share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. These observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes.It is known that proteins are far from equilibrium during folding reactions, and they undergo a wide range of conformational states to reach the global folding minimum. Various physical and chemical strategies, such as the use of high temperature, high pressure, protonation, altered ionic strength, and harsh denaturants, are commonly used to disturb folding species to promote the formation of rarely observed folding intermediates. From the thermodynamic point of view, any perturbing agent affecting protein folding is controlled by Le Chatelier’s principle. For instance, increasing the concentration of urea shifts the folding equilibrium toward the unfolded state because of the increased preferential binding of urea to this state. In the case of pressure, the smaller volume of the unfolded state is favored at high pressure because it only affects the volumetric properties of the molecule.The energy landscape theory of folding assumes that protein folding is the progressive organization of an ensemble of partially folded structures through which the protein passes on its way to a native conformation (1–3). Accordingly, proteins have a rugged funnel-like landscape that is biased toward their native structure due to evolution (3). Obtaining structural and dynamic information on multiple-stage protein intermediates that follow the folding trail may increase our knowledge of protein misfolding, which is associated with amyloidosis, prion formation, and the occurrence of several diseases, including Parkinson’s disease, Huntington’s disease, spongiform encephalopathy, and, more recently, cancer (4–6). In these diseases, proteins tend to traverse the “wrong side of the funnel” (7, 8).The effects of pressure on proteins were discovered in early experiments with egg albumin (9). Currently, pressure is extensively used in various biological and biotechnological applications (10–13) and is one of the most promising variables enabling the structural analysis of these protein substates. The application of pressure to a protein forces the water shell into the protein, thus shifting the equilibrium of the system from the native state to an intermediate or to the unfolded state. Pressure appears to favor water infiltration into the protein and the disassembly of protein cavities (14), leading to increased hydration and decreased partial molar volume. Unlike high temperatures, which cause systematic changes in the total energy and volume of the molecule, high pressure affects only the volumetric properties of the molecule. However, under physiological conditions, water solvation dictates protein folding (15).High pressure can be coupled with various spectroscopic techniques, including methods based on the intrinsic fluorescence of Trp residues (16), Fourier-transform infrared spectroscopy (FTIR) (17), NMR (13, 18, 19), small-angle X-ray scattering (SAXS) (20, 21), microsecond pressure-jump coupled to fluorescence lifetime (22), and, more recently, circular dichroism (CD) (23). To better understand local changes, high-pressure NMR (HP-NMR) is adopted as the most informative approach (18, 19). SAXS is another useful tool for assessing changes in protein folding and the size and shape of macromolecules in solution (24–26).Most denaturants affect protein folding through their binding properties. Urea is one of the most commonly used chemical denaturants for the study of protein folding and thermodynamics. Two main theories exist to explain why urea induces protein denaturation (27, 28). The first theory hypothesizes an indirect mechanism by which urea alters the water structure and leads to hydrophobic group solvation. The second view is based on the direct interaction of urea with the protein. Most studies are based on model compounds and theoretical and modeling approaches, such as molecular dynamics (29–35).The binding of urea to proteins is based on the ability of urea to displace water molecules from the first solvation shell (29), thus increasing the system entropy and weakening the hydrophobic effect (36). Pioneer calorimetric studies on protein–urea interactions were developed by Makhatadze and Privalov (37). Urea is also believed to form hydrogen bonds with the amide unit of peptide bonds, as shown by calorimetric measurements of cyclic dipeptides (36) and H/D exchange by 1D NMR of an alanine-based model compound (38). A two-stage kinetic mechanism for the action of urea has been proposed based on extensive (microsecond) molecular-dynamics simulations (29). However, at equilibrium conditions, experimental insights into the action of urea at the initial stages of denaturation are lacking. A long-standing controversy in the literature concerns the contribution of nonpolar groups, peptide backbone interactions, or both as the driving force behind urea-induced protein denaturation (32, 33, 39, 40). Experimental efforts were mostly based on measurements of transfer free energies (41, 42) of amino acid side chains, peptide backbones, or random-coil polypeptide chains, in which packing defects and the overall 3D architecture of a native polypeptide chain were neglected.Here, we studied Moniliophthora perniciosa necrosis- and ethylene-inducing protein 2 (MpNep2). We systematically studied protein folding in response to chemical (urea), physical (pressure), or both perturbations using HP-NMR spectroscopy and 3D low-resolution shape reconstructions from SAXS data to better understand how each of the two disturbing agents (pressure and urea) affect protein folding and to examine how urea denatures proteins. The use of these synergistic techniques (HP-NMR and SAXS) provides reliable structural information on local/global intermediates within protein energy landscapes. We provide evidence of a conformational state in which urea binds to the protein without promoting full denaturation by populating a dry molten globule (DMG). This state is more sensitive to pressure (lower p_1/2) due to the presence of a pulling force and an increased volume change.