Pressure-Induced Protein Unfolding in the Ternary System AOT−Octane−Water Is Different from that in Bulk Water

Abstract

In a cellular environment, the presence of macromolecular cosolutes and membrane interfaces can influence the folding-unfolding behavior of proteins. Here we report on the pressure stability of alpha-chymotrypsin in the ternary system bis(2-ethylhexyl)sodium sulfosuccinate-octane-water using FTIR spectroscopy. The ternary system forms anionic reverse micelles which mimic cellular conditions. We find that inclusion of a single protein molecule in a reverse micelle does not alter its conformation. When pressurized in bulk water, alpha-chymotrypsin unfolds at 750 MPa into a partially unfolded structure. In contrast, in the ternary system, the same pressure increase induces a random coil-like unfolded state, which collapses into an amorphous aggregate during the decompression phase. It is suggested that the unfolding pathway is different in a cell-mimicking environment due to the combined effect of multiple factors, including confinement. A phase transition of the reverse micellar to the lamellar phase is thought to be essential to provide the conditions required for unfolding and aggregation, though the unfolding is not a direct result of the phase transition. Our observations therefore suggest that membranes may cause the formation of alternative conformations that are more susceptible to aggregation.

Oxidative refolding of reduced, denatured lysozyme in AOT reverse micelles

“…Our data demonstrated that the refolding kinetics of lysozyme in reverse micelles were remarkably different from those in bulk water. A recent work has suggested that the unfolding pathway of α-chymotrypsin in AOT reverse micelles is different from that in bulk water due to the combined effects of multiple factors, including molecular confinement [22]. When pressurized in bulk water, α-chymotrypsin unfolds at 750 MPa into a partially unfolded structure.…”

Amyloid Fibril Nucleation in Reverse Micelles

“…Moreover, cellular environment is considered as -crowded‖ because it contains high concentration of soluble macromolecules that occupy between 10 to 40% of the total fluid volume of the cell (Fulton, 1982;Zimmerman & Trach, 1991). High concentrations of dissolved macromolecules that alter the bulk properties of water, and lead to changes in the structure of proteins and the activity of enzymes (Chang, Liu, & Chen, 1994;Cringus et al, 2007;Faeder & Ladanyi, 2000;Fayer, 2011;Fragoso, Pacheco, & Karmali, 2012;Meersman, Dirix, Shipovskov, Klyachko, & Heremans, 2005;Moilanen, Fenn, Wong, & Fayer, 2009;Mukherjee, Chowdhury, & Gai, 2007). The effects of confinement and crowding may be mimicked in reverse micelles (RMs), making them useful models of biological microenvironments (Fayer, 2011).…”

Infrared spectroscopy of proteins in reverse micelles

“…Further studies of α-chymotrypsin in CATB reverse micelles were performed by Celej et al, who found that encapsulation increased α-helix content, and decreased β-sheet content – correlating with increased enzymatic activity [4]. Meersman et al also examined α-chymotrypsin, but focused on pressure-induced unfolding in AOT reverse micelles, concluding that encapsulation significantly affected the unfolding pathway [32]. …”

“…These microenvironments are subject to macromolecular crowding, with high concentrations of dissolved substances that alter the bulk properties of water, and lead to changes in the structure of proteins and the activity of enzymes [5,10,13,18,32,34,35]. The effects of confinement and crowding may be mimicked in reverse micelles, making them useful models of biological microenvironments [15].…”