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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

Individual rates of folding and unfolding of the protease monomers and the observed relaxation rate for the concentration of 5 μM dimer.Both rates are fitted by linear functions. The crossing point roughly corresponds with the inflex point of the unfolding transition. Rate constants are considered as dimensionless quantities related to the units of h−1.
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pone.0119099.g009: Individual rates of folding and unfolding of the protease monomers and the observed relaxation rate for the concentration of 5 μM dimer.Both rates are fitted by linear functions. The crossing point roughly corresponds with the inflex point of the unfolding transition. Rate constants are considered as dimensionless quantities related to the units of h−1.

Mentions: The values of the relaxation rate constant kobs (p) were determined for all the pressures and the rate constants kf (p) and ku (p) were calculated using Eqs. (13–15). Fig. 9 shows linear dependences of the folding and unfolding rate constants, while the relaxation constant kobs (p) has a minimum in the crossing point of the former lines where the equilibrium constant equals 1. This feature indicates microreversibility of the transition. Finally, the apparent activation volume change and the atmospheric pressure rate constants kf,atm and ku,atm were determined using linear regression of the data by the functionslnki(p)=lnki,atm−ΔpΔVi≠RT,(21)where “I” stands for “u” or “f” and is the activation volume of the respective reaction. The values for 2 and 5 μM dimer are shown in Table 1. It is obvious that these values, especially the rate constants, are strongly pressure dependent which might be a consequence of the dimerization influence on the unfolding process. Thus, these parameters cannot be considered as reliable characteristic of the unfolding itself, but as an evidence of the mutual interplay of unfolding and dimerization.


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)

Individual rates of folding and unfolding of the protease monomers and the observed relaxation rate for the concentration of 5 μM dimer.Both rates are fitted by linear functions. The crossing point roughly corresponds with the inflex point of the unfolding transition. Rate constants are considered as dimensionless quantities related to the units of h−1.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119099.g009: Individual rates of folding and unfolding of the protease monomers and the observed relaxation rate for the concentration of 5 μM dimer.Both rates are fitted by linear functions. The crossing point roughly corresponds with the inflex point of the unfolding transition. Rate constants are considered as dimensionless quantities related to the units of h−1.
Mentions: The values of the relaxation rate constant kobs (p) were determined for all the pressures and the rate constants kf (p) and ku (p) were calculated using Eqs. (13–15). Fig. 9 shows linear dependences of the folding and unfolding rate constants, while the relaxation constant kobs (p) has a minimum in the crossing point of the former lines where the equilibrium constant equals 1. This feature indicates microreversibility of the transition. Finally, the apparent activation volume change and the atmospheric pressure rate constants kf,atm and ku,atm were determined using linear regression of the data by the functionslnki(p)=lnki,atm−ΔpΔVi≠RT,(21)where “I” stands for “u” or “f” and is the activation volume of the respective reaction. The values for 2 and 5 μM dimer are shown in Table 1. It is obvious that these values, especially the rate constants, are strongly pressure dependent which might be a consequence of the dimerization influence on the unfolding process. Thus, these parameters cannot be considered as reliable characteristic of the unfolding itself, but as an evidence of the mutual interplay of unfolding and dimerization.

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