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Inhibitor and substrate binding induced stability of HIV-1 protease against sequential dissociation and unfolding revealed by high pressure spectroscopy and kinetics.

Ingr M, Lange R, Halabalová V, Yehya A, Hrnčiřík J, Chevalier-Lucia D, Palmade L, Blayo C, Konvalinka J, Dumay E - PLoS ONE (2015)

Bottom Line: In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor.High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate.High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.

View Article: PubMed Central - PubMed

Affiliation: Tomas Bata University in Zlín, Faculty of Technology, Department of Physics and Material Engineering, nám. T.G. Masaryka 5555, 76001 Zlín, Czech Republic; Charles University in Prague, Department of Biochemistry, Hlavova 2030, 128 43 Prague 2, Czech Republic.

ABSTRACT
High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of -32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 μM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.

No MeSH data available.


Related in: MedlinePlus

Fluorescence-intensity indicated transition for 5 μM dimer.A. Time dependence of the intensity for different pressures. Each series is plotted together with the fitting single-exponential decay curve. The same sample was used for the whole set of measurements, pressure setting was facilitated by pressure jumps. The numbers indicate pressure in MPa. B. Equilibrium values of fluorescence for the same experimental series determined from the limit of the fitting curves for time tending to infinity. Inflex point is indicated by the open triangle.
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pone.0119099.g008: Fluorescence-intensity indicated transition for 5 μM dimer.A. Time dependence of the intensity for different pressures. Each series is plotted together with the fitting single-exponential decay curve. The same sample was used for the whole set of measurements, pressure setting was facilitated by pressure jumps. The numbers indicate pressure in MPa. B. Equilibrium values of fluorescence for the same experimental series determined from the limit of the fitting curves for time tending to infinity. Inflex point is indicated by the open triangle.

Mentions: In addition to CSM, spectral intensity of the measured spectra was studied, too. The curves representing the pressure dependence of the spectral integral look qualitatively similar to the CSM curves (see the non-inhibited curves in Fig. 1), but they vary for quite a long time period. Therefore, a series of kinetic experiments was carried out in order to determine the time dependence of the spectral intensity at different pressures and concentrations. The measured curves (Fig. 7) show an obvious decrease within the time course, the magnitude of which depends on the pressure. A series of pressure-jump experiments with varying enzyme concentrations was performed. A typical set of the measured intensity curves including the fitting single-exponential functions is presented in Fig. 8A. The pressure dependent fluorescence intensity changes were not fully reversible. However, at the end of each kinetics, no further time dependent changes occurred, allowing the determination of apparent thermodynamic parameters. The apparent equilibrium values of fluorescence were evaluated for each curve and plotted as functions of pressure (an example is given in Fig. 8B). These functions have a sigmoid-like profile typical for the structural transitions. The values of the apparent volume change ΔVu and atmospheric pressure Gibbs-energy change of this transition were determined by means of non-linear regression. They are listed in Table 1 and show relatively high stability with respect to the varying concentration, which indicates a transition of first-order kinetics in both directions. Thus, this transition represents presumably an unfolding of the protease monomers. of the series with the highest concentration is somewhat higher in comparison with the other concentrations which might be a consequence of preventing the unfolding by dimer formation.


Inhibitor and substrate binding induced stability of HIV-1 protease against sequential dissociation and unfolding revealed by high pressure spectroscopy and kinetics.

Ingr M, Lange R, Halabalová V, Yehya A, Hrnčiřík J, Chevalier-Lucia D, Palmade L, Blayo C, Konvalinka J, Dumay E - PLoS ONE (2015)

Fluorescence-intensity indicated transition for 5 μM dimer.A. Time dependence of the intensity for different pressures. Each series is plotted together with the fitting single-exponential decay curve. The same sample was used for the whole set of measurements, pressure setting was facilitated by pressure jumps. The numbers indicate pressure in MPa. B. Equilibrium values of fluorescence for the same experimental series determined from the limit of the fitting curves for time tending to infinity. Inflex point is indicated by the open triangle.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362767&req=5

pone.0119099.g008: Fluorescence-intensity indicated transition for 5 μM dimer.A. Time dependence of the intensity for different pressures. Each series is plotted together with the fitting single-exponential decay curve. The same sample was used for the whole set of measurements, pressure setting was facilitated by pressure jumps. The numbers indicate pressure in MPa. B. Equilibrium values of fluorescence for the same experimental series determined from the limit of the fitting curves for time tending to infinity. Inflex point is indicated by the open triangle.
Mentions: In addition to CSM, spectral intensity of the measured spectra was studied, too. The curves representing the pressure dependence of the spectral integral look qualitatively similar to the CSM curves (see the non-inhibited curves in Fig. 1), but they vary for quite a long time period. Therefore, a series of kinetic experiments was carried out in order to determine the time dependence of the spectral intensity at different pressures and concentrations. The measured curves (Fig. 7) show an obvious decrease within the time course, the magnitude of which depends on the pressure. A series of pressure-jump experiments with varying enzyme concentrations was performed. A typical set of the measured intensity curves including the fitting single-exponential functions is presented in Fig. 8A. The pressure dependent fluorescence intensity changes were not fully reversible. However, at the end of each kinetics, no further time dependent changes occurred, allowing the determination of apparent thermodynamic parameters. The apparent equilibrium values of fluorescence were evaluated for each curve and plotted as functions of pressure (an example is given in Fig. 8B). These functions have a sigmoid-like profile typical for the structural transitions. The values of the apparent volume change ΔVu and atmospheric pressure Gibbs-energy change of this transition were determined by means of non-linear regression. They are listed in Table 1 and show relatively high stability with respect to the varying concentration, which indicates a transition of first-order kinetics in both directions. Thus, this transition represents presumably an unfolding of the protease monomers. of the series with the highest concentration is somewhat higher in comparison with the other concentrations which might be a consequence of preventing the unfolding by dimer formation.

Bottom Line: In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor.High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate.High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.

View Article: PubMed Central - PubMed

Affiliation: Tomas Bata University in Zlín, Faculty of Technology, Department of Physics and Material Engineering, nám. T.G. Masaryka 5555, 76001 Zlín, Czech Republic; Charles University in Prague, Department of Biochemistry, Hlavova 2030, 128 43 Prague 2, Czech Republic.

ABSTRACT
High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of -32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 μM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.

No MeSH data available.


Related in: MedlinePlus