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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

Concentration dependence of the inflex point of the CSM curves.The data consist of two independent experimental series (blue and red circles). Linear-regression line of the slope of 76.3 MPa and intercept of 45 MPa is shown which is used to determine the constants Kd,atm = 0.92 μM and ΔVr = 32.5 ml mol−1. Regression lines of the individual series are not shown as they are almost identical; their slopes and intercepts are 77.3 MPa and 43 MPa, respectively (blue), and 74.7 MPa and 47 MPa, respectively (red), which corresponds with Kd,atm = 0.95 μM, ΔVr = 32.1 ml mol−1 (red) and Kd,atm = 0.88 μM, ΔVr = 33.2 ml mol−1 (blue). The error bars of the individual measurements are calculated from the errors of the regression parameters of Eq. 19 using Eq. 20 and the error-transition law. [M0] is considered to be a dimensionless quantity related to the units of μM ([M0] → [M0]/μM).
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pone.0119099.g005: Concentration dependence of the inflex point of the CSM curves.The data consist of two independent experimental series (blue and red circles). Linear-regression line of the slope of 76.3 MPa and intercept of 45 MPa is shown which is used to determine the constants Kd,atm = 0.92 μM and ΔVr = 32.5 ml mol−1. Regression lines of the individual series are not shown as they are almost identical; their slopes and intercepts are 77.3 MPa and 43 MPa, respectively (blue), and 74.7 MPa and 47 MPa, respectively (red), which corresponds with Kd,atm = 0.95 μM, ΔVr = 32.1 ml mol−1 (red) and Kd,atm = 0.88 μM, ΔVr = 33.2 ml mol−1 (blue). The error bars of the individual measurements are calculated from the errors of the regression parameters of Eq. 19 using Eq. 20 and the error-transition law. [M0] is considered to be a dimensionless quantity related to the units of μM ([M0] → [M0]/μM).

Mentions: The inflex points of the CSM curves apparently shift to higher pressures with increasing concentration, which is characteristic for transitions where the reaction volume decreases along with dissociation of multisubunit complexes, e.g. dimer dissociation. In addition, the limits of the curves at plus and minus infinity have non-zero, but practically equal slope corresponding to that of the inhibited curves. The curves of the two highest concentrations turn steeply up above 350 MPa, likely due to the protein aggregation (see below). This part of the curves was therefore excluded from the evaluation of the dimerization parameters. Inflex points of all the curves were determined and plotted vs. logarithm of monomer concentration, as shown in Fig. 5. Dimer-dissociation volume change ΔVd = (−32.5 ± 4.1)ml mol−1 and the atmospheric-pressure equilibrium constant Kd,atm = (0.92 ± 0.17)μM were evaluated by means of linear regression in accord with Eq. (8). The ratio of the volume change to molar weight of HIV-1 PR (−1.50 ml/kg) is in a reasonable agreement with previously studied proteins yeast enolase (−1.28 ml/kg) [20] or yeast hexokinase (from −1.07 to −1.49 ml/kg) [21].


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)

Concentration dependence of the inflex point of the CSM curves.The data consist of two independent experimental series (blue and red circles). Linear-regression line of the slope of 76.3 MPa and intercept of 45 MPa is shown which is used to determine the constants Kd,atm = 0.92 μM and ΔVr = 32.5 ml mol−1. Regression lines of the individual series are not shown as they are almost identical; their slopes and intercepts are 77.3 MPa and 43 MPa, respectively (blue), and 74.7 MPa and 47 MPa, respectively (red), which corresponds with Kd,atm = 0.95 μM, ΔVr = 32.1 ml mol−1 (red) and Kd,atm = 0.88 μM, ΔVr = 33.2 ml mol−1 (blue). The error bars of the individual measurements are calculated from the errors of the regression parameters of Eq. 19 using Eq. 20 and the error-transition law. [M0] is considered to be a dimensionless quantity related to the units of μM ([M0] → [M0]/μM).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119099.g005: Concentration dependence of the inflex point of the CSM curves.The data consist of two independent experimental series (blue and red circles). Linear-regression line of the slope of 76.3 MPa and intercept of 45 MPa is shown which is used to determine the constants Kd,atm = 0.92 μM and ΔVr = 32.5 ml mol−1. Regression lines of the individual series are not shown as they are almost identical; their slopes and intercepts are 77.3 MPa and 43 MPa, respectively (blue), and 74.7 MPa and 47 MPa, respectively (red), which corresponds with Kd,atm = 0.95 μM, ΔVr = 32.1 ml mol−1 (red) and Kd,atm = 0.88 μM, ΔVr = 33.2 ml mol−1 (blue). The error bars of the individual measurements are calculated from the errors of the regression parameters of Eq. 19 using Eq. 20 and the error-transition law. [M0] is considered to be a dimensionless quantity related to the units of μM ([M0] → [M0]/μM).
Mentions: The inflex points of the CSM curves apparently shift to higher pressures with increasing concentration, which is characteristic for transitions where the reaction volume decreases along with dissociation of multisubunit complexes, e.g. dimer dissociation. In addition, the limits of the curves at plus and minus infinity have non-zero, but practically equal slope corresponding to that of the inhibited curves. The curves of the two highest concentrations turn steeply up above 350 MPa, likely due to the protein aggregation (see below). This part of the curves was therefore excluded from the evaluation of the dimerization parameters. Inflex points of all the curves were determined and plotted vs. logarithm of monomer concentration, as shown in Fig. 5. Dimer-dissociation volume change ΔVd = (−32.5 ± 4.1)ml mol−1 and the atmospheric-pressure equilibrium constant Kd,atm = (0.92 ± 0.17)μM were evaluated by means of linear regression in accord with Eq. (8). The ratio of the volume change to molar weight of HIV-1 PR (−1.50 ml/kg) is in a reasonable agreement with previously studied proteins yeast enolase (−1.28 ml/kg) [20] or yeast hexokinase (from −1.07 to −1.49 ml/kg) [21].

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