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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

Equilibrium values of CSM of ANS emission spectrum for 10 μM dimer fitted by a model function.Values for the reverse course of pressure indicate irreversibility of the transition.
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pone.0119099.g010: Equilibrium values of CSM of ANS emission spectrum for 10 μM dimer fitted by a model function.Values for the reverse course of pressure indicate irreversibility of the transition.

Mentions: 8-anilino-1-naphthalenesulfonic acid (ANS) is a fluorescent indicator that binds to proteins, mainly by hydrophobic interactions. This interaction is accompanied with a fluorescence increase and a blue shift of ANS emission spectrum, which can be used to indicate the structural changes of proteins, especially the unfolding [34]. A series of fluorescence spectra under various pressures was measured as described in Methods and Material. Up to 240 MPa only a continuous decrease of the CSM with growing pressure occurs without any sign of a transition. Above this pressure, a remarkable change in the spectral shape takes place due to the increased emission at 472 nm which shifts CSM steeply up. The time course of this transition was investigated by pressure-jump experiments. A time series of emission spectra was taken at each pressure point and the pressure dependence of CSM was fitted by Eq. (19). The apparent equilibrium values were plotted vs. pressure (Fig. 10) and the transition parameters were determined as in the previous section. The values of apparent ΔGANS,atm = (43.5 ± 5.8)kJ mol−1 and ΔVANS = (−160 ± 21)ml mol−1 confirm a transition strongly unfavourable at pressures up to 240 MPa with atmospheric-pressure equilibrium constant KANS,atm = 2.4 × 10−8, but highly favourable above 320 MPa. Above this pressure the shift of equilibrium CSM value is small indicating that the transition runs almost to completeness. Stepwise release of the pressure did not lead to the return of the fluorescence intensity to the value before the transition. Hence, this transition is either irreversible or the kinetics of the reverse process is very slow.


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)

Equilibrium values of CSM of ANS emission spectrum for 10 μM dimer fitted by a model function.Values for the reverse course of pressure indicate irreversibility of the transition.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119099.g010: Equilibrium values of CSM of ANS emission spectrum for 10 μM dimer fitted by a model function.Values for the reverse course of pressure indicate irreversibility of the transition.
Mentions: 8-anilino-1-naphthalenesulfonic acid (ANS) is a fluorescent indicator that binds to proteins, mainly by hydrophobic interactions. This interaction is accompanied with a fluorescence increase and a blue shift of ANS emission spectrum, which can be used to indicate the structural changes of proteins, especially the unfolding [34]. A series of fluorescence spectra under various pressures was measured as described in Methods and Material. Up to 240 MPa only a continuous decrease of the CSM with growing pressure occurs without any sign of a transition. Above this pressure, a remarkable change in the spectral shape takes place due to the increased emission at 472 nm which shifts CSM steeply up. The time course of this transition was investigated by pressure-jump experiments. A time series of emission spectra was taken at each pressure point and the pressure dependence of CSM was fitted by Eq. (19). The apparent equilibrium values were plotted vs. pressure (Fig. 10) and the transition parameters were determined as in the previous section. The values of apparent ΔGANS,atm = (43.5 ± 5.8)kJ mol−1 and ΔVANS = (−160 ± 21)ml mol−1 confirm a transition strongly unfavourable at pressures up to 240 MPa with atmospheric-pressure equilibrium constant KANS,atm = 2.4 × 10−8, but highly favourable above 320 MPa. Above this pressure the shift of equilibrium CSM value is small indicating that the transition runs almost to completeness. Stepwise release of the pressure did not lead to the return of the fluorescence intensity to the value before the transition. Hence, this transition is either irreversible or the kinetics of the reverse process is very slow.

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