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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

Spectral properties of inhibited (solid circles) and non-inhibited (open circles) HIV-1 PR as functions of pressure.A. CSM of the tryptophan fluorescence spectrum, λex = 280 nm, λem = 300–400 nm. Curves are mutually shifted by an additive constant in order to allow the comparison of their shape details. Solid lines represent the regression curves of the data series (linear for inhibited and sigmoidal according to Eq.19 for non-inhibited curve) and dashed lines the corresponding confidence bands. As in Fig. 3, points of the non-inhibited series above 375 MPa, which are influenced by aggregation, are excluded from the regression procedure. B. Spectral integral of the same spectra.
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pone.0119099.g001: Spectral properties of inhibited (solid circles) and non-inhibited (open circles) HIV-1 PR as functions of pressure.A. CSM of the tryptophan fluorescence spectrum, λex = 280 nm, λem = 300–400 nm. Curves are mutually shifted by an additive constant in order to allow the comparison of their shape details. Solid lines represent the regression curves of the data series (linear for inhibited and sigmoidal according to Eq.19 for non-inhibited curve) and dashed lines the corresponding confidence bands. As in Fig. 3, points of the non-inhibited series above 375 MPa, which are influenced by aggregation, are excluded from the regression procedure. B. Spectral integral of the same spectra.

Mentions: HIV-1 protease solution of 12 μM dimer was mixed with 120 μM darunavir and the pressure was increased stepwise from the lowest value of 10 MPa to the maximum of 500 MPa recording the fluorescence spectrum in every pressure point. Fig. 1 shows the comparison of pressure dependences of CSM and spectral integral for inhibited and non-inhibited enzyme. It can be seen that the spectral integral change of the inhibited enzyme is relatively small in comparison with the strong sigmoid-like decrease of the free-enzyme curve. Accordingly, the CSM curve of the inhibited-enzyme decreases almost linearly, while the free-enzyme curve follows this trend only at the beginning of the pressure range up to approximately 200 MPa and then diverges from the inhibited curve to lower values. This trend is then reversed at 375 MPa, from where the curve goes steeply up. The continuous CSM decrease common for both the curves is likely an expression of a direct influence of the high pressure to the fluorophores as was demonstrated by [31] on free tryptophan and its derivatives. Its slope is equal for both the curves in the pressure range where the free enzyme can be expected to stay in the unperturbed dimeric form. Hence, the “inhibited” curve shows neither the characteristic sigmoidal shape indicating a conformation transition nor any other substantial variations of the trend, while the “free” curve does. Thus, both CSM and spectral intensity indicate that the structure of the enzyme is stabilized by the inhibitor within the chosen pressure range. This conclusion is supported also by the differential spectra shown in S2 Fig. (see the next section).


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)

Spectral properties of inhibited (solid circles) and non-inhibited (open circles) HIV-1 PR as functions of pressure.A. CSM of the tryptophan fluorescence spectrum, λex = 280 nm, λem = 300–400 nm. Curves are mutually shifted by an additive constant in order to allow the comparison of their shape details. Solid lines represent the regression curves of the data series (linear for inhibited and sigmoidal according to Eq.19 for non-inhibited curve) and dashed lines the corresponding confidence bands. As in Fig. 3, points of the non-inhibited series above 375 MPa, which are influenced by aggregation, are excluded from the regression procedure. B. Spectral integral of the same spectra.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119099.g001: Spectral properties of inhibited (solid circles) and non-inhibited (open circles) HIV-1 PR as functions of pressure.A. CSM of the tryptophan fluorescence spectrum, λex = 280 nm, λem = 300–400 nm. Curves are mutually shifted by an additive constant in order to allow the comparison of their shape details. Solid lines represent the regression curves of the data series (linear for inhibited and sigmoidal according to Eq.19 for non-inhibited curve) and dashed lines the corresponding confidence bands. As in Fig. 3, points of the non-inhibited series above 375 MPa, which are influenced by aggregation, are excluded from the regression procedure. B. Spectral integral of the same spectra.
Mentions: HIV-1 protease solution of 12 μM dimer was mixed with 120 μM darunavir and the pressure was increased stepwise from the lowest value of 10 MPa to the maximum of 500 MPa recording the fluorescence spectrum in every pressure point. Fig. 1 shows the comparison of pressure dependences of CSM and spectral integral for inhibited and non-inhibited enzyme. It can be seen that the spectral integral change of the inhibited enzyme is relatively small in comparison with the strong sigmoid-like decrease of the free-enzyme curve. Accordingly, the CSM curve of the inhibited-enzyme decreases almost linearly, while the free-enzyme curve follows this trend only at the beginning of the pressure range up to approximately 200 MPa and then diverges from the inhibited curve to lower values. This trend is then reversed at 375 MPa, from where the curve goes steeply up. The continuous CSM decrease common for both the curves is likely an expression of a direct influence of the high pressure to the fluorophores as was demonstrated by [31] on free tryptophan and its derivatives. Its slope is equal for both the curves in the pressure range where the free enzyme can be expected to stay in the unperturbed dimeric form. Hence, the “inhibited” curve shows neither the characteristic sigmoidal shape indicating a conformation transition nor any other substantial variations of the trend, while the “free” curve does. Thus, both CSM and spectral intensity indicate that the structure of the enzyme is stabilized by the inhibitor within the chosen pressure range. This conclusion is supported also by the differential spectra shown in S2 Fig. (see the next section).

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