Remodeling of the Folding Free Energy Landscape of Staphylococcal Nuclease by Cavity-Creating Mutations

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 proteinurea 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.protein folding | hydrostatic pressure | urea | NMR | SAXS

Section: Measuring Urea Effects Based On Nmr Peak Intensities and Saxssupporting

confidence: 60%

A hypothesis to reconcile the physical and chemical unfolding of proteins

Oliveira

Silva

2015

“…In particular, we are interested in the structural origins of the volume changes that underlie the shift in equilibrium between folded conformations at moderate pressures, rather than those that lead to a pressuredenatured state (14)(15)(16)(17)(18)(19)(20), or to an unfolded state formed at high pressure in the presence of a chemical denaturant (21,52). In the present study unfolding will refer to a process in which loss of tertiary and secondary structure occurs.…”

Section: Discussionmentioning

confidence: 99%

Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure

Lerch

López

Yang

et al. 2015

Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306-E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electronelectron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ∼3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111-114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2-3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape.DEER | EPR | conformational exchange | protein structural dynamics P roteins in solution exist in conformational equilibria that cannot be appreciated from structures observed in crystal lattices (1-5). The members of a folded conformational ensemble may have distinct functions and hence are of interest in elucidating mechanisms of protein action (5-7). The free-energy differences between the conformations can range from zero to a few kilocalories per mole; the higher free-energy states are referred to as "invisible" or "excited" (E) states owing to their low equilibrium populations. The structural transition between the native ground state (G) and the E state may involve rigid body motion of the peptide backbone (5, 8) or local unfolding (9).For a complete understanding of molecular mechanisms underlying function, characterization of functionally relevant conformational substates is required. However, in the case of E states, low populations and short lifetimes present a challenge for biophysical characterization. The elegant high-resolution NMR studies from Akasaka (10, 11) and coworkers suggest that application of hydrostatic pressure on the order of a few kilobars may solve this problem by reversibly populating functional E states, making them amenable for study by spectroscopic methods. For example, high-pressure NMR has been used to identify and characterize E states crucial to ligand binding in ubiquitin (12) and d...

“…A more mechanical view is that pressure-induced unfolding is essentially a result of the relief of packing defects in the native conformations of proteins (3,5,51). Recent studies of cavity mutants of SNase, in which a correlation between the site-resolved volume changes upon unfolding and void volumes within the native state was observed, reinforce this interpretation (4,5,52). The SNase results provide compelling evidence that the reflection of Le Chatelier's principle in the elimination of void spaces within the folded state can dominate pressure-induced unfolding.…”

Section: Discussionmentioning

confidence: 99%

“…The underlying determinants of pressure-induced unfolding have recently been a subject of several detailed investigations (2)(3)(4)(5)(6)(7)(8)(9)(10). Fundamentally, pressure-induced unfolding of proteins results from the population of nonnative conformations having a lower total system volume than the native structure seen at ambient pressure.…”

mentioning

confidence: 99%

“…With the development of highpressure sample cells compatible with modern solution NMR probes (15), detailed measurements of proteins unfolding under pressure with atomic resolution have now become possible (5,(16)(17)(18). Recent studies of staphylococcal nuclease (SNase) compellingly argue that the filling of void volumes present in the native state is the primary determinant of pressure-induced unfolding (4)(5)(6). A critical aspect of a "destruction of voids" mechanism for pressure-induced unfolding of proteins is whether the voids or cavities are occupied with water in the folded state.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Role of cavities and hydration in the pressure unfolding of T₄lysozyme

Nucci

Fuglestad

Athanasoula

et al. 2014

It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T 4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A-benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T 4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.protein stability | protein folding and cooperativity | protein hydration | high-pressure NMR | reverse micelle encapsulation