Pressure-Induced Protein Unfolding in the Ternary System AOT-Octane-Water Is Different from that in Bulk Water Filip Meersman, †, * Carolien Dirix, § Stepan Shipovskov, ¶ Natalia L. Klyachko, | and Karel Heremans §, * Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200D, B-3001 Leuven, Belgium, Department of Molecular Biophysics, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden, and Department of Chemical Enzymology, Faculty of Chemistry, Moscow State University, 119899 Moscow, Russia Received December 1, 2004. In Final Form: January 31, 2005 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 R-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,R-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. Introduction Most biochemical reactions, such as protein folding and unfolding, are studied in vitro in dilute aqueous solutions. However, in vivo these reactions occur in a highly crowded environment.Macromolecular crowding willaffect the equilibria and rates of these reactions. 1-3 In addition, there is the presence of lipid membranes which interact with many proteins,whether these are membrane-bound or not. 4,5 It has been demonstrated that membranes can influence the folding-unfolding reactions of proteins and change the protein conformation compared to the solution state. 6,7 Of particular interest is the possible role of lipid membranes in a number of human diseases such as Creutzfeldt-Jakob disease, light chain amyloidosis, and Alzheimer’s disease. 8-11 These diseases are characterized by the deposition of threadlike,ordered proteinaceous aggregates called amyloid fibrils. It has been demonstrated for several disease-related proteins that their assembly into fibrillar structures can be catalyzed by anionic membranes. 9,12,13 In this work, we explore the pressure-induced unfolding of R-chymotrypsin in the ternary system bis(2-ethylhexyl)- sodium sulfosuccinate (AOT)-octane-water. This system spontaneously forms reverse micelles, which are water- in-oil droplets consisting of spheroidal assemblies of AOT whereby the polar headgroup ofthe AOT molecule is directed toward the water phase in the interior of the sphere and the hydrocarbon tails are in contact with the bulk organic solvent. The AOT layer can be considered a membrane mimic, thereby providing a water-membrane interface. 14-16 Moreover, this ternary system has two additional advantagescompared to other membrane mimics such as sodium dodecyl sulfate micelles and lipid vesicles, which are oil-in-water systems. Because the size of reverse micelles depends solely on the molar ratio of water to surfactant (w o ), it is possible to create conditions in which a single protein molecule surrounded by a water shell is incorporated in a reverse micelle. As such, reverse micelles represent (i) a confined geometry similar to that induced by intracellular crowding, 17 as well as (ii) a low * Authors to whom correspondence should be addressed. Tel: 0032 16 32 71 59 (K.H.); 0044 1223 76 38 45 (F.M.). Fax: 0032 16 32 79 82 (K.H.); 0044 1223 76 38 49 (F.M.). E-mail: karel.heremans@fys.kuleuven.ac.be (K.H.); fpsm2@cam.ac.uk (F.M.). † University of Cambridge. § Katholieke Universiteit Leuven. ¶ Lund University. | Moscow State University. (1) van den Berg, B.; Ellis, R. J.; Dobson, C. M. EMBO J. 1999, 18, 6927. (2) Minton, A. P. Curr. Opin. Struct. Biol. 2000, 10, 34. (3) Ellis, R. J. Trends Biochem. Sci. 2001, 26, 597. (4) Epand, R. M. Biochim. Biophys. Acta 1998, 1376, 353. (5) Klyachko, N. L.; Levashov, P. 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