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.

Charles University Prague Department of Biochemistry Hlavova 2030 128 43 Prague 2 Czech Republic

INSERM U710 Université Montpellier 2 Place Eugene Bataillon 34095 Montpellier cedex 05 France

Tomas Bata University in Zlín Faculty of Technology Department of Physics and Material Engineering nám T G Masaryka 5555 76001 Zlín Czech Republic

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 Prague Department of Biochemistry Hlavova 2030 128 43 Prague 2 Czech Republic

Université Montpellier 2 INRA UMR IATE Biochimie et Technologie Alimentaires cc023 Place Eugene Bataillon 34095 Montpellier cedex 05 France

Université Montpellier 2 UMR IATE Biochimie et Technologie Alimentaires cc023 2 Place Eugene Bataillon 34095 Montpellier cedex 05 France

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Precursors of Viral Proteases as Distinct Drug Targets

Inhibition of the precursor and mature forms of HIV-1 protease as a tool for drug evaluation