High pressure modulates amyloid formation.

A common mechanism of conformational changes and pathological aggregation of proteins associated with amyloid diseases remains to be proven. High pressure is emerging as a new strategy for studying aspects of amyloid formation. Pressure provides a convenient means to populate and characterize partially folded states, which are thought to have a key role in assembly processes of proteins into amyloid fibrils. High pressure can also be used to dissociate aggregates and amyloid fibrils or on the opposite to generate such species.

Pressure and temperature as tools for investigating the role of individual non-covalent interactions in enzymatic reactions Sulfolobus solfataricus carboxypeptidase as a model enzyme.

Sulfolobus solfataricus carboxypeptidase, (CPSso), is a heat- and pressure-resistant zinc-metalloprotease. Thanks to its properties, it is an ideal tool for investigating the role of non-covalent interactions in substrate binding. It has a broad substrate specificity as it can cleave any N-blocked amino acid (except for N-blocked proline). Its catalytic and kinetic mechanisms are well understood, and the hydrolytic reaction is easily detectable spectrophotometrically. Here, we report investigations on the pressure- and temperature-dependence of the kinetic parameters (turnover number and Michaelis constant) of CPSso using several benzoyl- and 3-(2-furyl)acryloyl-amino acids as substrates. This approach enabled us to study these parameters in terms of individual rate constants and establish that the release of the free amino acid is the rate-limiting step, making it possible to dissect the individual non-covalent interactions participating in substrate binding. In keeping with molecular docking experiments performed on the 3D model of CPSso available to date, our results show that both hydrophobic and energetic interactions (i.e., stacking and van der Waals) are mainly involved, but their contribution varies strongly, probably due to changes in the conformational state of the enzyme.

What lies in the future of high-pressure bioscience?

Without being comprehensive in this mini-review, I will address perspectives, some speculative, for the development and use of high pressure to explore biochemical phenomena. This will be illustrated with several examples.

Linear and non-linear pressure dependence of enzyme catalytic parameters.

The pressure dependence of enzyme catalytic parameters allows volume changes associated with substrate binding and activation volumes for the chemical steps to be determined. Because catalytic constants are composite parameters, elementary volume change contributions can be calculated from the pressure differentiation of kinetic constants. Linear and non-linear pressure-dependence of single-step enzyme reactions and steady-state catalytic parameters can be observed. Non-linearity can be interpreted either in terms of interdependence between the pressure and other environmental parameters (i.e., temperature, solvent composition, pH), pressure-induced enzyme unfolding, compressibility changes and pressure-induced rate limiting changes. These different situations are illustrated with several examples.

The role of the 132-160 region in prion protein conformational transitions.

The native conformation of host-encoded cellular prion protein (PrP(C)) is metastable. As a result of a post-translational event, PrP(C) can convert to the scrapie form (PrP(Sc)), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown. However, several studies have highlighted the importance of the N-terminal region spanning residues 90-154 in PrP folding. In order to understand why PrP folds into two different conformational states exhibiting distinct secondary and tertiary structure, and to gain insight into the involvement of this particular region in PrP transconformation, we studied the pressure-induced unfolding/ refolding of recombinant Syrian hamster PrP expanding from residues 90-231, and compared it with heat unfolding. By using two intrinsic fluorescent variants of this protein (Y150W and F141W), conformational changes confined to the 132-160 segment were monitored. Multiple conformational states of the Trp variants, characterized by their spectroscopic properties (fluorescence and UV absorbance in the fourth derivative mode), were achieved by tuning the experimental conditions of pressure and temperature. Further insight into unexplored conformational states of the prion protein, likely to mimic the in vivo structural change, was obtained from pressure-assisted cold unfolding. Furthermore, salt-induced conformational changes suggested a structural stabilizing role of Tyr150 and Phe141 residues, slowing down the conversion to a beta-sheet form.

Pressure equilibrium and jump study on unfolding of 23-kDa protein from spinach photosystem II.

Pressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20 degrees C and pH 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (DeltaG(u)) and volume change (DeltaV(u)) for the unfolding are 5.9 kcal/mol and -160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in DeltaG(u) but also with a decrease in DeltaV(u). The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (-66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.

Use of hydrostatic pressure to produce 'native' monomers of yeast enolase.

The effects of hydrostatic pressure on yeast enolase have been studied in the presence of 1 mm Mn(2+). When compared with apo-enolase, and Mg-enolase, the Mn-enzyme differs from the others in three ways. Exposure to hydrostatic pressure does not inactivate the enzyme. If the experiments are performed in the presence of 1 mm Mg(2+), or with apo-enzyme, the enzyme is inactivated [Kornblatt, M.J., Lange R., Balny C. (1998) Eur. J. Biochem 251, 775-780]. The UV spectra of the high pressure forms of the Mg(2+)- and apo-forms of enolase are identical and distinct from the spectrum of the form obtained in the presence of 1 mm Mn(2+); this suggests that Mn(2+) remains bound to the high pressure form of enolase. With Mn-enolase, the various spectral changes do not occur in the same pressure range, indicating that multiple processes are occurring. Pressure experiments were performed as a function of [Mn(2+)] and [protein]. One of the changes in the UV spectra shows a dependence on protein concentration, indicating that enolase is dissociating into monomers. The small changes in the UV spectrum and the retention of activity lead to a model in which enolase, in the presence of high concentrations of Mn(2+), dissociates into native monomers; upon release of pressure, the enzyme is fully active. Although further spectral changes occur at higher pressures, there is no inactivation as long as Mn(2+) remains bound. We propose that the relatively small and polar nature of the subunit interface of yeast enolase, including the presence of several salt bridges, is responsible for the ability of hydrostatic pressure to dissociate this enzyme into monomers with a native-like structure.

High pressure induces scrapie-like prion protein misfolding and amyloid fibril formation.

Our understanding of conformational conversion of proteins in diseases is essential for any diagnostic and therapeutic approach. Although not fully understood, misfolding of the prion protein (PrP) is implicated in the pathogenesis of prion diseases. Despite several efforts to produce the pathologically misfolded conformation in vitro from a recombinant PrP, no positive result has yet been obtained. Within the "protein-only hypothesis", the reason for this hindrance may be that the experimental conditions used did not allow selection of the pathway adopted in vivo resulting in conversion into the infectious form. Here, using a pressure perturbation approach, we show that recombinant PrP is converted to a novel misfolded conformer, which is prone to aggregate and ultimately form amyloid fibrils. A short incubation at high pressure (600 MPa) of the truncated form of hamster prion protein (SHaPrP(90-231)) resulted in the formation of pre-amyloid structures. The mostly globular aggregates were characterized by ThT and ANS binding, and by a beta-sheet-rich secondary structure. After overnight incubation at 600 MPa, amyloid fibrils were formed. In contrast to pre-amyloid structures, they showed birefringency of polarized light after Congo red staining and a strongly decreased ANS binding capacity, but enhanced ThT binding. Both aggregate types were resistant to digestion by PK, and can be considered as potential scrapie-like forms or precursors. These results may be useful for the search for compounds preventing pathogenic PrP misfolding and aggregation.

Rate-determining step of butyrylcholinesterase-catalyzed hydrolysis of benzoylcholine and benzoylthiocholine. Volumetric study of wild-type and D70G mutant behavior.

The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BuChE) is deacylation (k(3)), whereas it is acylation (k(2)) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BuChE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown. Nonlinearity of the plots may be explained in terms of compressibility changes or rate-limiting changes. To distinguish between these two possibilities, enzyme phosphorylation by diisopropylfluorophosphate (DFP) in the presence of substrate (BzSCh) under pressure was studied. There was no pressure dependence of volume changes for DFP binding or for phosphorylation of either wild-type or D70G. Analysis of the pressure dependence for steady-state hydrolysis of substrates, and for phosphorylation by DFP provided evidence that no enzyme compressibility changes occurred during the catalyzed reactions. Thus, the nonlinear pressure dependence of substrate hydrolysis reflects changes in the rate-limiting step with pressure. Change in rate-determining step occurred at a pressure of 100 MPa for hydrolysis of BzCh by wild-type and at 75 MPa for D70G. For hydrolysis of BzSCh the change occurred at higher pressures because k(2) << k(3) at atmospheric pressure for this substrate. Elementary volume change contributions upon initial binding, productive binding, acylation and deacylation were calculated from the pressure differentiation of kinetic constants. This analysis shed light on the molecular events taking place along the hydrolysis pathways of BzCh and BzSCh by wild-type BuChE and the D70G mutant. In addition, volume change differences between wild-type and D70G provided new evidence that residue D70 in the peripheral site controls hydration of the active site gorge and the dynamics of the water molecule network during catalysis. Finally, a steady-state kinetic study of the oxyanion hole mutant (G117H) showed that substitution of the ethereal sulfur for oxygen in the substrate alters the final adjustment of substrate in the active site and stabilization of the acylation transition state.

The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure.

The heat- and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20-75 degrees C or the pressure range of 0.1-700 MPa. Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety. In contrast, the mature form, which lacks the propeptide composed of 91 amino acid residues, showed more complex behaviour: differential scanning calorimetry and pressure-induced changes in fluorescence were consistent with a three-step transition. These results show that carboxypeptidase Y is composed of two structural domains, which unfold independently but that procarboxypeptidase Y behaves as a single domain, thus ensuring cooperative unfolding. The carbohydrate moiety has a slightly protective role in heat-induced unfolding and a highly protective role in pressure-induced unfolding.

Formation of transient oxygen complexes of cytochrome p450 BM3 and nitric oxide synthase under high pressure.

The kinetics of formation and transformation of oxygen complexes of two heme-thiolate proteins (the F393H mutant of cytochrome P450 BM3 and the oxygenase domain of endothelial nitric oxide synthase, eNOS) were studied under high pressure. For BM3, oxygen-binding characteristics (rate and activation volume) matched those measured for CO-binding. In contrast, pressure revealed a different CO- and oxygen-binding mechanism for eNOS, suggesting that it is hazardous to take CO-binding as a model for oxygen-binding. With eNOS, a ferric NO complex is formed as an intermediate in the second reaction cycle. Here we report the pressure stability of this compound. Furthermore, in the presence of 4-amino-tetrahydrobiopterin (ABH(4)), an analog to the natural second electron donor tetrahydrobiopterin (BH(4)), biphasic pressure profiles of the oxygen-binding rates were observed, both in the first and the second reaction cycles, indicative of the formation of an additional reaction intermediate. This was confirmed by experiments where ABH(4) was replaced by ABH(2), a cofactor which cannot deliver an electron. Altogether, high pressure appears to be a useful tool to characterize elementary steps in the reaction cycle of heme-thiolate proteins.

[Thermodynamic and kinetic analysis of unfolding of P23k protein isolated from spinach photosystem II].

The unfolding of 23kD (P23k) protein isolated from spinach photosystem II particle was studied by high pressure and fluorescence spectroscopy. The thermal equilibrium study indicated that the protein could be totally unfolded by 180 or 160 MPa at 20 degrees C and 3 degrees C, respectively. The standard free energy and standard volume change of the protein for unfolding at 20 degrees C is 23.45 kJ/mol and -150.3 ml/mol, respectively. Kinetics study indicated that at 20 degrees C the activation volume for unfolding, delta V(u)(++), was negative (-66.2 ml/mol), meanwhile the activation volume for folding, deltaV(f)(++), was positive (84.1 ml/mol). The rate constants for folding and unfolding (K(0f), K(0u)) were 1.87 s(-1) and 1.3x10(-4) s(-1), respectively, these results provide some clues to explain why the protein is so sensitive to pressure.

The fate of the prion protein in the prion/plasminogen complex.

The cellular prion protein (PrP(c)) forms complexes with plasminogen. Here, we show that the PrP(c) in this complex is cleaved to yield fragments of PrP(c). The cleavage is accelerated by plasmin but does not appear to be dependent on it.

The thermodynamic analysis of protein stabilization by sucrose and glycerol against pressure-induced unfolding.

We have studied the reaction native left arrow over right arrow denatured for the 33-kDa protein isolated from photosystem II. Sucrose and glycerol have profound effects on pressure-induced unfolding. The additives shift the equilibrium to the left; they also cause a significant decrease in the standard volume change (DeltaV). The change in DeltaV was related to the sucrose and glycerol concentrations. The decrease in DeltaV varied with the additive: sucrose caused the largest effect, glycerol the smallest. The theoretical shift of the half-unfolding pressure (P1/2) calculated from the net increase in free energy by addition of sucrose and glycerol was lower than that obtained from experimental mea- surements. This indicates that the free energy change caused by preferential hydration of the protein is not the unique factor involved in the protein stabilization. The reduction in DeltaV showed a large contribution to the theoretical P1/2 shift, suggesting that the DeltaV change, caused by the sucrose or glycerol was associated with the protein stabilization. The origin of the DeltaV change is discussed. The rate of pressure-induced unfolding in the presence of sucrose or glycerol was slower than the refolding rate although both were significantly slower than that observed without any stabilizers.

Optimized overproduction, purification, characterization and high-pressure sensitivity of the prion protein in the native (PrP(C)-like) or amyloid (PrP(Sc)-like) conformation.

Overproduction and purification of the prion protein is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. We have developed a method for the effective cloning, overexpression in Escherichia coli and purification to homogeneity of Syrian golden hamster prion protein (SHaPrP(90-231)). A high level of overexpression, resulting in the formation of inclusion bodies, was obtained under the control of the T7-inducible promoter of the pET15b plasmid. The protein required denaturation, reduction and refolding steps to become soluble and attain its native conformation. Purification was carried out by differential centrifugation, gel filtration and reverse phase chromatography. An improved cysteine oxidation protocol using oxidized glutathione under denaturing conditions, resulted in the recovery of a higher yield of chromatographically pure protein. About 10 mg of PrP protein per liter of bacterial culture was obtained. The recombinant protein was identified by monoclonal antibodies and its integrity was confirmed by electrospray mass spectrometry (ES/MS), whereas correct folding was assessed by circular dichroism (CD) spectroscopy. This protein had the structural characteristics of PrP(C) and could be converted to an amyloid structure sharing biophysical and biochemical properties of the pathologic form (PrP(Sc)). The sensitivity of these two forms to high pressure was investigated. We demonstrate the potential of using pressure as a thermodynamic parameter to rescue trapped aggregated prion conformations into a soluble state, and to explore new conformational coordinates of the prion protein conformational landscape.

Alternative prion structural changes revealed by high pressure.

At high temperature, recombinant hamster prion protein (SHaPrP(90-231)) undergoes aggregation and changes from a predominantly alpha-helical to beta-sheet conformation. We then applied high pressure (200 MPa) to the beta-sheet-rich conformation. The aggregation was reversed, and the original tertiary and secondary structures were recovered at ambient pressure, after pressure release. The application of a pressure of 200 MPa thus allowed studying the heat-induced equilibrium refolding in the absence of protein aggregation. Prion protein unfolding as a function of high pressure was also investigated. Simple two-state, reversible unfolding transitions were observed, as monitored by spectral changes in the UV and fluorescence of the hydrophobic probe 8-anilino-1-naphthalene sulfonate. However, these heat- and pressure-induced conformers differed in their unfolding free energy. At pressures over 400 MPa, strong thioflavin-T binding was observed, suggesting a further structural change to a metastable oligomeric structure.

Fluorescence and FTIR study of pressure-induced structural modifications of horse liver alcohol dehydrogenase (HLADH).

The process of pressure-induced modification of horse liver alcohol dehydrogenase (HLADH) was followed by measuring in situ catalytic activity (up to 250 MPa), intrinsic fluorescence (0.1-600 MPa) and modifications of FTIR spectra (up to 1000 MPa). The tryptophan fluorescence measurements and the kinetic data indicated that the pressure-induced denaturation of HLADH was a process involving several transitions and that the observed transient states have characteristic properties of molten globules. Low pressure (< 100 MPa) induced no important modification in the catalytic efficiency of the enzyme and slight conformational changes, characterized by a small decrease in the centre of spectral mass of the enzyme's intrinsic fluorescence: a native-like state was assumed. Higher pressures (100-400 MPa) induced a strong decrease of HLADH catalytic efficiency and further conformational changes. At 400 MPa, a dimeric molten globule-like state was proposed. Further increase of pressure (400-600 MPa) seemed to induce the dissociation of the dimer leading to a transition from the first dimeric molten globule state to a second monomeric molten globule. The existence of two independent structural domains in HLADH was assumed to explain this transition: these domains were supposed to have different stabilities against high pressure-induced denaturation. FTIR spectroscopy was used to follow the changes in HLADH secondary structures. This technique confirmed that the intermediate states have a low degree of unfolding and that no completely denatured form seemed to be reached, even up to 1000 MPa.

A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II.

An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.

Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate: analysis of volume changes upon reaction and hysteretic behavior.

Hydrolysis of the neutral substrate N-methylindoxyl acetate (NMIA) by wild-type human butyrylcholinesterase (BuChE) and peripheral site mutants (D70G, Y332A, D70G/Y332A) was found to follow the Michaelis-Menten kinetics. K(m) was 0.14 mM for wild-type, and 0.07-0.16 mM for D70G, Y332A and D70G/Y332A, indicating that the peripheral site is not involved in NMIA binding. The values of k(cat) were of the same order for all enzymes: 12,000-18,000 min(-1). Volume changes upon substrate binding (-DeltaV(K(m))) and the activation volumes (DeltaV++(k(cat)) associated with hydrolysis of NMIA were calculated from the pressure dependence of the catalytic constants. Values of -DeltaV(K(m)) indicate that NMIA binds to an aromatic residue, presumed to be W82, the active site binding locus. Binding is accompanied by a release of water molecules from the gorge. Residue 70 controls the number of water molecules that are released upon substrate binding. The values of DeltaV++(k(cat)), which are positive for wild-type and faintly positive for D70G, clearly indicate that the catalytic steps are accompanied by re-entry of water into the gorge. Results support the premise that residue D70 is involved in the conformational stabilization of the active site gorge and in control of its hydration. A slow transient, preceding the steady state, was seen on a time scale of several minutes. The induction time rapidly increased with NMIA concentration to reach a limit at substrate saturation. Much shorter induction times (<1 min) were seen for hydrolysis of benzoylcholine (BzCh) by wild-type BuChE and for hydrolysis of butyrylthiocholine (BuSCh) by the active site mutants E197Q and E197Q/G117H. This slow transient was interpreted in terms of hysteresis without kinetic cooperativity. The hysteretic behavior of BuChE results from a slow conformational equilibrium between two enzyme states E and E'. NMIA binds only to the primed form E'. Kosmotropic salts and hydrostatic pressure were found to shift the equilibrium toward E'. The E-->E' transition is accompanied by a negative activation volume (DeltaV++(0)= -45+/-10 ml/mol), and the E' form is more compact than E. Hydration water in the gorge of E' appears to be more structured than in the unprimed form.