Origins of Pressure-Induced Protein Transitions

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...

Section: Discussionmentioning

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

Lerch

López

Yang

et al. 2015

“…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. Various mechanisms for pressure-induced unfolding have been proposed including changes in water structure that weaken the hydrophobic effect at high pressure (11,12), increases in solvent density at the protein surface that contribute to a reduction in the total volume of the protein-water system (13,14), and the elimination of cavities in the protein interior through exposure to solvent (3). 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).…”

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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

“…Analysis of high-resolution X-ray structures suggested that formation of intramolecular hydrogen bonds and van der Waals interactions in the native state lead to a decrease in atomic volumes and thus to a decrease in protein volume upon folding (5). Further, water around hydrophobic groups has a larger volume than bulk water, which also leads to a decrease in volume upon burial of hydrophobic groups in the native protein (6). On the other hand, formation of ordered water structures around charged groups (7) and solvation of the peptide backbone decrease the water volume (6), which leads to a volume increase upon folding.…”

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Transition state and ground state properties of the helix–coil transition in peptides deduced from high-pressure studies

Neumaier

Büttner

Bachmann

et al. 2013

Volume changes associated with protein folding reactions contain valuable information about the folding mechanism and the nature of the transition state. However, meaningful interpretation of such data requires that overall volume changes be deconvoluted into individual contributions from different structural components. Here we focus on one type of structural element, the α-helix, and measure triplet-triplet energy transfer at high pressure to determine volume changes associated with the helix-coil transition. Our results reveal that the volume of a 21-amino-acid alanine-based peptide shrinks upon helix formation. Thus, helices, in contrast with native proteins, become more stable with increasing pressure, explaining the frequently observed helical structures in pressureunfolded proteins. Both helix folding and unfolding become slower with increasing pressure. per molecule) for removing one residue. Thus, addition or removal of a helical residue proceeds through a transitory high-energy state with a large volume, possibly due to the presence of unsatisfied hydrogen bonds, although steric effects may also contribute.protein stability | helix dynamics | protein dynamics U nderstanding the effect of pressure on protein stability and dynamics provides insight into fundamental principles and mechanisms of protein folding (1). For most proteins, increasing pressure shifts the folding equilibrium toward the unfolded state, which, according to Le Chatelier's principle, shows that the native state has a larger volume than the unfolded state (2-4). The origin of the volume increase upon folding has been discussed controversially for a long time. The major obstacle in the interpretation of volume changes is opposing contributions from different effects. Analysis of high-resolution X-ray structures suggested that formation of intramolecular hydrogen bonds and van der Waals interactions in the native state lead to a decrease in atomic volumes and thus to a decrease in protein volume upon folding (5). Further, water around hydrophobic groups has a larger volume than bulk water, which also leads to a decrease in volume upon burial of hydrophobic groups in the native protein (6). On the other hand, formation of ordered water structures around charged groups (7) and solvation of the peptide backbone decrease the water volume (6), which leads to a volume increase upon folding. Recent experimental results suggested that volume changes associated with transfer of groups from solvent to the protein interior upon folding are small and that the formation of void volumes in native proteins is the major origin of the volume increase upon folding (8).Volume changes associated with the formation of protein folding transition states are only poorly characterized. Highpressure stopped-flow experiments on tendamistat (9) and cold shock protein (10), as well as pressure-jump experiments on coldshock protein (10) and an ankyrin repeat domain (11), revealed that volumes of protein folding transition states are close to the volume of the native...