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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

Initial rate of substrate cleavage as a function of pressure related to the value for 10 MPa.Different symbols denote three experimental series. The solid curve shows the relative rate for the ideal case of pressure dependent Kd but pressure independent Km and kcat.
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pone.0119099.g011: Initial rate of substrate cleavage as a function of pressure related to the value for 10 MPa.Different symbols denote three experimental series. The solid curve shows the relative rate for the ideal case of pressure dependent Kd but pressure independent Km and kcat.

Mentions: Enzyme kinetics was investigated under the varying pressure in the region of 10–350 MPa. The substrate was mixed in a spectrophotometric cuvette with the reaction buffer and enzyme and this sample was transferred immediately to the high-pressure cell of the instrument. As this manipulation requires time in the order of seconds to minutes, the concentrations were chosen in order that the linear decrease of substrate concentration takes considerably longer time. The measured reaction rate was then normalised to the initial value at 10 MPa and plotted vs. pressure (see Fig. 11). The resulting curve is decreasing steeply but the activity remains detectable up to 350 MPa. The identification of the individual phenomena contributing to this decrease is rather difficult. The general function describing the rate of the enzyme reaction, which can be derived from the ordinary Michaelis-Menten kinetics and the monomer-dimer equilibrium (Eq. 3), isv=kcatSKm+S[D0+KmKd8(Km+S)(1−1+16D0(Km+S)KmKd)].(22)


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)

Initial rate of substrate cleavage as a function of pressure related to the value for 10 MPa.Different symbols denote three experimental series. The solid curve shows the relative rate for the ideal case of pressure dependent Kd but pressure independent Km and kcat.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119099.g011: Initial rate of substrate cleavage as a function of pressure related to the value for 10 MPa.Different symbols denote three experimental series. The solid curve shows the relative rate for the ideal case of pressure dependent Kd but pressure independent Km and kcat.
Mentions: Enzyme kinetics was investigated under the varying pressure in the region of 10–350 MPa. The substrate was mixed in a spectrophotometric cuvette with the reaction buffer and enzyme and this sample was transferred immediately to the high-pressure cell of the instrument. As this manipulation requires time in the order of seconds to minutes, the concentrations were chosen in order that the linear decrease of substrate concentration takes considerably longer time. The measured reaction rate was then normalised to the initial value at 10 MPa and plotted vs. pressure (see Fig. 11). The resulting curve is decreasing steeply but the activity remains detectable up to 350 MPa. The identification of the individual phenomena contributing to this decrease is rather difficult. The general function describing the rate of the enzyme reaction, which can be derived from the ordinary Michaelis-Menten kinetics and the monomer-dimer equilibrium (Eq. 3), isv=kcatSKm+S[D0+KmKd8(Km+S)(1−1+16D0(Km+S)KmKd)].(22)

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