Pressure‐induced molten globule state of cholinesterase

Cléry, Cécile; Renault, Frédérique; Masson, Patrick

doi:10.1016/0014-5793(95)00787-a

Cited by 55 publications

(38 citation statements)

References 25 publications

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“…This finding is in agreement with experimental observations (13), most notably the observed increase in the hydrodynamic radius upon denaturation (48,49) and an increase in the hydrogen-exchange rates of lysozyme and RNase A with pressure (50). This swelling process results in structures with reduced compactness that, however, retain considerably more order than heat-denatured proteins, as probed by NMR experiments of hydrogen exchange (17).…”

Section: Discussionsupporting

confidence: 82%

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.

572

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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Section: Discussionsupporting

confidence: 82%

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.

572

516

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“…High pressure has been found to promote formation of molten globule intermediates (48)(49)(50)(51)(52)(53). Although it is customary to think of molten globules as highly aggregation-competent conformations, high pressure appears to inhibit this tendency.…”

Section: Discussionmentioning

confidence: 99%

High pressure fosters protein refolding from aggregates at high concentrations

John

Carpenter

Randolph

1999

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

157

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High hydrostatic pressures (1-2 kbar), combined with low, nondenaturing concentrations of guanidine hydrochloride (GdmHCl) foster disaggregation and refolding of denatured and aggregated human growth hormone and lysozyme, and ␤-lactamase inclusion bodies. One hundred percent recovery of properly folded protein can be obtained by applying pressures of 2 kbar to suspensions containing aggregates of recombinant human growth hormone (up to 8.7 mg͞ml) and 0.75 M GdmHCl. Covalently crosslinked, insoluble aggregates of lysozyme could be refolded to native, functional protein at a 70% yield, independent of protein concentration up to 2 mg͞ml. Inclusion bodies containing ␤-lactamase could be refolded at high yields of active protein, even without added GdmHCl.

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“…The conformation of E2 trapped under high pressure is more compact than the urea-denatured state, and it retains the ability to bind bis-ANS, features compatible with molten globule-like conformations (43,44). High pressure has been used to trap molten-globule conformations of different protein systems (15,24,45,46).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Partially Folded Monomer of the DNA-binding Domain of Human Papillomavirus E2 Protein Obtained at High Pressure

Foguel

Silva

Prat‐Gay³

1998

Journal of Biological Chemistry

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The pressure-induced dissociation of the dimeric DNA binding domain of the E2 protein of human papillomavirus (E2-DBD) is a reversible process with a K d of 5.6 ؋ 10 ؊8 M at pH 5.5. The complete exposure of the intersubunit tryptophans to water, together with the concentration dependence of the pressure effect, is indicative of dissociation. Dissociation is accompanied by a decrease in volume of 76 ml/mol, which corresponds to an estimated increase in solvent-exposed area of 2775 Å 2 . There is a decrease in fluorescence polarization of tryptophan overlapping the red shift of fluorescence emission, supporting the idea that dissociation of E2-DBD occurs in parallel with major changes in the tertiary structure. The dimer binds bis(8-anilinonaphthalene-1-sulfonate), and pressure reduces the binding by about 30%, in contrast with the almost complete loss of dye binding in the urea-unfolded state. These results strongly suggest the persistence of substantial residual structure in the high pressure state. Further unfolding of the high pressure state was produced by low concentrations of urea, as evidenced by the complete loss of bis(8-anilinonaphthalene-1-sulfonate) binding with less than 1 M urea. Following pressure dissociation, a partially folded state is also apparent from the distribution of excited state lifetimes of tryptophan. The combined data show that the tryptophans of the protein in the pressure-dissociated state are exposed long enough to undergo solvent relaxation, but the persistence of structure is evident from the observed internal quenching, which is absent in the completely unfolded state. The average rotational relaxation time (derived from polarization and lifetime data) of the pressure-induced monomer is shorter than the urea-denatured state, suggesting that the species obtained under pressure are more compact than that unfolded by urea.

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Pressure‐induced molten globule state of cholinesterase

Cited by 55 publications

References 25 publications

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

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

High pressure fosters protein refolding from aggregates at high concentrations

Characterization of a Partially Folded Monomer of the DNA-binding Domain of Human Papillomavirus E2 Protein Obtained at High Pressure

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