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.

Evolution under Drug Pressure Remodels the Folding Free-Energy Landscape of Mature HIV-1 Protease

“…These unfolding experiments conducted at a lower protein concentration allowed us to determine both the K dimer at atmospheric pressure and the volume change associated with the change of K dimer with pressure, the apparent Δ V diss . A K dimer of 1.4 ± 0.2 μM was estimated from these experiments, in very good agreement with previously reported value of 1.3 μM for PR D25N 26 , together with a Δ V diss of −12 ± 4 ml/mol, which is significantly smaller than the Δ V diss =−32.5 ml/mol recently reported by Ingr et al 39 . The difference in the sample used (PR D25N instead of PR) and the experimental method (NMR instead of Trp fluorescence) likely explain the discrepancies in the estimated Δ V diss values.…”

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

“…Studies have shown that the PR precursor has a much lower tendency to form dimers than mature PR 13 , 14 , and it shows much lower activity and possibly modified specificity 15 – 17 . On the other hand, it is likely stabilized by substrate binding 18 . Viral p6* protein, located directly upstream of the PR domain (Fig.…”

Equilibria of oligomeric proteins under high pressure – A theoretical description

“…High-pressure methods are used to investigate protein denaturation, unfolding, conformational changes, enzyme kinetics, etc., but they also have valuable application in studying quaternary structure and equilibria of oligomeric proteins. Many oligomeric proteins have been investigated by high-pressure methods, including those of low number of subunits, mainly dimers (Paladini and Weber, 1981;Silva et al, 1986;Ruan and Weber, 1988;Erijman et al, 1993;Kornblatt et al, 2004;Marchal et al, 2012;Ingr et al, 2015) and tetramers (Jaenicke and Koberstein, 1971;Royer et al, 1986;Ruan and Weber, 1989;Pin et al, 1990;Devillebonne and Else, 1991; Ruan and Weber, 1993;Girard et al, 2010), hexamers (Foguel and Weber, 1995), higher oligomers and viral capsids (Silva and Weber, 1988;Silva et al, 1989Silva et al, , 1992Da Poian et al, 1993;Silva et al, 1996;Weber et al, 1996) or prion oligomers (Torrent et al, 2015), protein aggregates with less organized structure like casein micelles (Gebhardt et al, 2005(Gebhardt et al, , 2006(Gebhardt et al, , 2011 and even polymeric structures, e.g. TMV-virus (Bonafe et al, 1998) or microtubules and microfilaments (Messier and Seguin, 1978;Kobori et al, 1996;Nishiyama et al, 2010).…”

“…Structural changes of oligomeric proteins prior to subunit dissociation were studied, too (Cioni and Strambini, 1996). These studies were concerned with different structural and functional features and in some cases the key thermodynamic parameters, especially the volume change of the oligomer dissociation V  and the atmospheric pressure equilibrium constant atm K , were determined (Silva et al, 1986;Weber, 1988, 1989;Pin et al, 1990;Dapoian et al, 1993;Erijman et al, 1993;Ruan and Weber, 1993;Foguel and Weber, 1995;Kornblatt et al, 2004;Ingr et al, 2015). These studies exploit the fact that the high pressure favors a process accompanied with a negative change of the total volume of the system.…”

“…Application of high pressure can induce various structural changes from oligomer dissociation via reversible unfolding to an irreversible aggregation, sometimes observable at a single protein (Dumay et al, 1994;Seefeldt et al, 2005;Ingr et al, 2015). It is, therefore, necessary to be able to distinguish among these processes, especially unfolding and oligomer dissociation.…”