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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

Time development of the tryptophan-fluorescence CSM.A. Time series for different pressures, each started with a new enzyme sample, concentration of 10 μM dimer. The individual curves are shifted up or down by artificially chosen constant for the sake of better orientation in the graph. B. Time series measured with the same sample of 2 μM concentration, pressure setting is facilitated by pressure jumps. The CSM scale is genuine for all the series.
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pone.0119099.g004: Time development of the tryptophan-fluorescence CSM.A. Time series for different pressures, each started with a new enzyme sample, concentration of 10 μM dimer. The individual curves are shifted up or down by artificially chosen constant for the sake of better orientation in the graph. B. Time series measured with the same sample of 2 μM concentration, pressure setting is facilitated by pressure jumps. The CSM scale is genuine for all the series.

Mentions: To confirm that the CSM at a given pressure does not change with time, two different kinetics experiments were carried out. In the first one a sample of the enzyme was put into the pressure cell and the pressure was set manually to the desired value from the range of 200 to 450 MPa. Then the tryptophan emission spectra were taken in a series of time points and the CSMs were determined. Fig. 4A shows that no significant CSM change can be observed at any of these curves. Up to 360 MPa slightly decreasing tendency can eventually be seen the magnitude of which is considerably lower than the CSM changes induced by the pressure increase. More significant increase can be observed at very high pressures above 400 MPa which can be associated with aggregation of the protein (see the description of this phenomenon below).


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)

Time development of the tryptophan-fluorescence CSM.A. Time series for different pressures, each started with a new enzyme sample, concentration of 10 μM dimer. The individual curves are shifted up or down by artificially chosen constant for the sake of better orientation in the graph. B. Time series measured with the same sample of 2 μM concentration, pressure setting is facilitated by pressure jumps. The CSM scale is genuine for all the series.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119099.g004: Time development of the tryptophan-fluorescence CSM.A. Time series for different pressures, each started with a new enzyme sample, concentration of 10 μM dimer. The individual curves are shifted up or down by artificially chosen constant for the sake of better orientation in the graph. B. Time series measured with the same sample of 2 μM concentration, pressure setting is facilitated by pressure jumps. The CSM scale is genuine for all the series.
Mentions: To confirm that the CSM at a given pressure does not change with time, two different kinetics experiments were carried out. In the first one a sample of the enzyme was put into the pressure cell and the pressure was set manually to the desired value from the range of 200 to 450 MPa. Then the tryptophan emission spectra were taken in a series of time points and the CSMs were determined. Fig. 4A shows that no significant CSM change can be observed at any of these curves. Up to 360 MPa slightly decreasing tendency can eventually be seen the magnitude of which is considerably lower than the CSM changes induced by the pressure increase. More significant increase can be observed at very high pressures above 400 MPa which can be associated with aggregation of the protein (see the description of this phenomenon below).

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