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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

By Marek Ingr, Reinhard Lange, Věra Halabalová, Alaa Yehya, Josef Hrnčiřík, Dominique Chevalier-Lucia, Laetitia Palmade, Claire Blayo, Jan Konvalinka and Eliane Dumay


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. © 2015 Ingr et al.INSERM; Grant Agency of the Czech Republic [P208-12-G016

Publisher: Public Library of Science
Year: 2015
DOI identifier: 10.1371/journal.pone.0119099
OAI identifier:

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