High-Pressure Denaturation of Staphylococcal Nuclease Proline-to-Glycine Substitution Mutants

Vidugiris, Gediminas; Truckses, Dagmar M.; Markley, John L.; Royer, Catherine A.

doi:10.1021/bi952012g

Cited by 50 publications

(41 citation statements)

References 40 publications

(61 reference statements)

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“…However, more recent experimental data (Vidugiris et al, 1996) indicated that the AV~n,l,ing values for H124L and H124L+P117G are identical within experimental error. We calculated the solvent-excluded volumes of H124L and H124L+P117G in the native (X-ray structures) and unfolded (modeled by extended chain) states using the PQMS program by Connolly (1985) ( …”

Section: Lys 116mentioning

confidence: 89%

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease

Truckses¹,

Somoza²,

Prehoda³

et al. 1996

Protein Science

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The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains HisIz4, is 1.9 kcal.molp' less stable (at pH 5.5 and 20 "C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains LeuIZ4. In addition, the population of the trans conformer at the Ly~'"-Pro''~ peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease HI 24L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and HI241 variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys'"-Pro'17 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys"h-Pro"7. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro' l7 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H 124L, and compared it with the published high-resolution X-ray structure of the cis form of the least stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the transkis equilibrium of the Lys'16-Pro'17 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination techniques. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L+P117G and H124L+P47G+P117G and the kinetic parameters of H124L, H124L+P47G, H124L+P117G, and H124L+P47G+P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kc,, affected more than Km, and their effects are additive. The PI 17G mutation in nuclease H124L leads to the same local conformational rearrangement described for the PI 17G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 335021-5030). In both P117G mutants, the loop formed by residues 112-1 17 is located closer to the adjacent loop formed by residues 77-...

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Section: Lys 116mentioning

confidence: 89%

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease

Truckses¹,

Somoza²,

Prehoda³

et al. 1996

Protein Science

Self Cite

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“…Activation volumes ⌬v u ‡ and ⌬v f ‡ are derived from a linear fit for pressures between 0 and 500 MPa. (1998) unfolding observed in pressure-jump experiments (41,57,58) suggest that sub-nanosecond molecular-dynamics simulations of pressure denaturation (59-62) do not fully cover the relevant time scales, although an increase in the solvent exposure of residues in the hydrophobic core has indeed been observed in an 800-ps simulation calculation (62). We compared observed activation volumes for folding and unfolding (41) and calculated activation volumes for forming and breaking hydrophobic contacts.…”

Section: Discussionmentioning

confidence: 99%

The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins

Hummer

Garde

Garcı́a

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

573

516

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Proteins can be denatured by pressures of a few hundred MPa. This finding apparently contradicts the most widely used model of protein stability, where the formation of a hydrophobic core drives protein folding. The pressure denaturation puzzle is resolved by focusing on the pressure-dependent transfer of water into the protein interior, in contrast to the transfer of nonpolar residues into water, the approach commonly taken in models of protein unfolding. Pressure denaturation of proteins can then be explained by the pressure destabilization of hydrophobic aggregates by using an information theory model of hydrophobic interactions. Pressure-denatured proteins, unlike heat-denatured proteins, retain a compact structure with water molecules penetrating their core. Activation volumes for hydrophobic contributions to protein folding and unfolding kinetics are positive. Clathrate hydrates are predicted to form by virtually the same mechanism that drives pressure denaturation of proteins.A decade ago, Walter Kauzmann (1) challenged the commonly held view that a hydrophobic core stabilizes globular proteins, by poignantly remarking that the ''liquid-hydrocarbon model (2) fails almost completely when one attempts to extend it to the effects of pressure on protein folding.'' Although a variety of forces stabilize folded proteins (3-6), the formation of a hydrophobic core is thought to play a dominant role. This view is supported by the temperature dependence of hydrophobic contributions to protein unfolding showing remarkable similarities to the transfer of hydrocarbons from a nonpolar phase into water, notably a convergence of the entropy of transfer (2,7,8). However, Kauzmann (1) pointed out that the pressure dependence of protein unfolding is at odds with the hydrophobic-core model: The volume change ⌬V upon unfolding is positive at low pressures but negative at pressures of about 100-200 MPa. The transfer of hydrocarbons into water shows exactly the opposite behavior, with ⌬V being negative at low pressures and positive at high pressures.Evidently, pressure unfolding of a protein (9-16) does not correspond to the transfer of a nonpolar molecule from a nonpolar environment into aqueous solution. Unlike heatdenatured proteins, the ensemble of pressure-denatured proteins retains elements of structural organization (13, 17). Consequently, an understanding of the thermodynamics of pressure denaturation might focus on the free energy of water transfer into the hydrophobic core of the protein (18) rather than transfer of nonpolar solutes into water. Our conceptual framework for pressure denaturation is as follows: the protein interior is largely composed of efficiently packed residues, more likely hydrophobic than those at the surface (19). Increasing hydrostatic pressure then forces water molecules into the protein interior, gradually filling cavities, and eventually breaking the protein structure apart.We therefore study the effects of pressure on the association of nonpolar residues in water. We use the informat...

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“…In this model, voids are eliminated by pressure owing to an increase in the population of an alternative packing arrangement of the core in which cavities are filled with native side chains rather than solvent. This model has been suggested to play a role in certain proteins at high pressure (1,(29)(30)(31), although to our knowledge direct observation of structure relaxation under pressure has not been reported.…”

mentioning

confidence: 86%

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

Lerch

López

Yang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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...

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High-Pressure Denaturation of Staphylococcal Nuclease Proline-to-Glycine Substitution Mutants

Cited by 50 publications

References 40 publications

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease

The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins

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

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