title: Inhibitor and substrate binding induced stability of HIV-1 protease against sequential dissociation and unfolding revealed by high pressure spectroscopy and kinetics.
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.
authors.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.
authors.forename: Marek
authors.initials: M
authors.lastname: Ingr
authors.affiliation: Université Montpellier 2, INRA UMR IATE, Biochimie et Technologie Alimentaires, cc023, Place Eugene Bataillon, 34095 Montpellier cedex 05, France.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lange
authors.affiliation: Tomas Bata University in Zlín, Faculty of Technology, Department of Physics and Material Engineering, nám. T.G. Masaryka 5555, 76001 Zlín, Czech Republic.
authors.forename: Věra
authors.initials: V
authors.lastname: Halabalová
authors.affiliation: INSERM U710, Université Montpellier 2, Place Eugene Bataillon, 34095 Montpellier cedex 05, France.
authors.forename: Alaa
authors.initials: A
authors.lastname: Yehya
authors.affiliation: Tomas Bata University in Zlín, Faculty of Technology, Department of Physics and Material Engineering, nám. T.G. Masaryka 5555, 76001 Zlín, Czech Republic.
authors.forename: Josef
authors.initials: J
authors.lastname: Hrnčiřík
authors.affiliation: Université Montpellier 2, UMR IATE, Biochimie et Technologie Alimentaires, cc023, 2 Place Eugene Bataillon, 34095 Montpellier cedex 05, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Chevalier-Lucia
authors.affiliation: Université Montpellier 2, UMR IATE, Biochimie et Technologie Alimentaires, cc023, 2 Place Eugene Bataillon, 34095 Montpellier cedex 05, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Palmade
authors.affiliation: Université Montpellier 2, UMR IATE, Biochimie et Technologie Alimentaires, cc023, 2 Place Eugene Bataillon, 34095 Montpellier cedex 05, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Blayo
authors.affiliation: Charles University in Prague, Department of Biochemistry, Hlavova 2030, 128 43 Prague 2, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Konvalinka
authors.affiliation: Université Montpellier 2, UMR IATE, Biochimie et Technologie Alimentaires, cc023, 2 Place Eugene Bataillon, 34095 Montpellier cedex 05, France.
authors.forename: Eliane
authors.initials: E
authors.lastname: Dumay
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001274:Atmospheric Pressure; D000069454:Darunavir / Q000378:metabolism*; D019281:Dimerization; D016333:HIV Protease / Q000737:chemistry* / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability / Q000187:drug effects*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000378:metabolism
chemical_list: D000817:Anilino Naphthalenesulfonates; D017320:HIV Protease Inhibitors; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1; D000069454:Darunavir