11118 DOI: 10.1021/la100891x Langmuir 2010, 26(13), 11118–11126 Published on Web 06/15/2010 pubs.acs.org/Langmuir © 2010 American Chemical Society Water Replacement Hypothesis in Atomic Details: Effect of Trehalose on the Structure of Single Dehydrated POPC Bilayers E. A. Golovina, † A. Golovin, ‡ F. A. Hoekstra, † and R. Faller* ,§ † Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands, ‡ Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow, Russia, and § Department of Chemical Engineering & Materials Science, University of California, Davis, Davis, California 95616 Received March 3, 2010. Revised Manuscript Received June 4, 2010 We present molecular dynamics (MD) simulations to study the plausibility of the water replacement hypothesis (WRH) from the viewpoint of structural chemistry. A total of 256 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) lipids were modeled for 400 ns at 11.7 or 5.4 waters/lipid. To obtain a single dehydrated bilayer relevant to the WRH, simulations were performed in the NP xy h z T ensemble with h z > 8 nm, allowing interactions between lipids in the membrane plane and preventing interactions between neighboring membranes via periodic boundary conditions. This setup resulted in a stable single bilayer in (or near) the gel state. Trehalose caused a concentration-dependent increase of the area per lipid (APL) accompanied by fluidizing the bilayer core. This mechanism has been suggested by the WRH. However, dehydrated bilayers in the presence of trehalose were not structurally identical to fully hydrated bilayers. The headgroup vector was in a more parallel orientation in dehydrated bilayers with respect to the bilayer plane and main- tained this orientation in the presence of trehalose in spite of APL increase. The total dipole potential changed sign in dehydrated bilayers and remained slightly positive in the presence of trehalose. The model of a dehydrated bilayer presented here allows the study of the mechanisms of membrane protection against desiccation by different compounds. Introduction It is well established that trehalose protects membranes in desic- cation tolerant organisms. 1,2 This “lesson from nature” is used to protect the content of dry liposomes against leakage in the phar- maceutical industry. 3,4 Trehalose can also ensure survival of human blood cells during freeze-drying, 5 which bears promise for blood banking. The water replacement hypothesis (WRH) describes the mecha- nism of membrane protection by trehalose. 1,2 This mechanism is based on replacement of water molecules by sugars in their inter- actions with polar groups of membrane lipids. These interactions maintain spacing between lipids and prevent the increase of the membrane main gel to fluid phase transition temperature T m . As a consequence, dry membranes remain in a fluid state at physio- logical temperatures and avoid a phase transition during rehy- dration. The transient coexistence of fluid and gel phases in a membrane during rehydration causes leakage and is detrimental for living organisms. The WRH has considerable experimental support (see e.g. refs 2 and 3 and references therein). However, all experimental data are indirect, and there is only limited structural data on lipids that can support mechanisms described by WRH. 6,7 Thus, alter- native hypotheses which explain experimental data by other mechanisms than interactions of disaccharides with lipid polar groups have been proposed. 8-11 They deny the role of sugar/lipid interactions and consider sugar vitrification as the main mechan- ism of membrane protection by trehalose at low (<20%) water content. Vitrification of the intermembrane layer causes the mem- branes to remain in the phase they were in at the time of vitrifi- cation. Therefore, if vitrification of the environment in the vicinity of membranes occurs when membranes are in a fluid state, they remain in this state in spite of dehydration. 9 The preferential exclu- sion theory of Timasheff 12 and the water entrapment hypothesis 13 were developed for protein protection at dehydration stress. Accord- ing to Timasheff, sugars are excluded from the vicinity of proteins, thus preserving their hydration shell and maintaining the neces- sary level of hydration during osmotic stress. This theory is valid for osmotic stress but does not relate to severe dehydration. Belton and Gil 13 extended the theory to dehydrated conditions and called it water-entrapment hypothesis. According to this hypothesis, water, *To whom correspondence should be addressed. (1) Crowe, J. H.; Crowe, L. M.; Chapman, D. Preservation of membranes in anhydrobiotic organisms - The role of trehalose. Science 1984, 223 (4637), 701- 703. (2) Crowe, J. H.; Hoekstra, F. A.; Crowe, L. M. Anhydrobiosis. Annu. Rev. Physiol. 1992, 54, 579-599. (3) Crowe, J. H.; Crowe, L. M.; Oliver, A. E.; Tsvetkova, N.; Wolkers, W.; Tablin, F. The trehalose myth revisited: Introduction to a symposium on stabilization of cells in the dry state. Cryobiology 2001, 43 (2), 89-105. (4) Crowe, J. H.; Crowe, L. M.; Wolkers, W. F.; Oliver, A. E.; Ma, X.; Auh, J.-H.; Tang, M.; Zhu, S.; Norris, J.; Tablin, F. Stabilization of Dry Mammalian Cells: Lessons from Nature. Integr. Comp. Biol. 2005, 45, 810-820. (5) Wolkers, W. F.; Walker, N. J.; Tablin, F.; Crowe, J. H. Human platelets loaded with trehalose survive freeze-drying. Cryobiology 2001, 42, 79-87. (6) Lee, C. W. B.; Das Gupta, S. K.; Mattai, J.; Shipley, G. G.; Abdel-Mageed, O. J.; Makriyannis, A.; Griffin, R. G. Characterization of the L-lambda phase in trehalose-stabilized dry membranes by solid-state NMR and X-ray diffraction. Biochemistry 1989, 28, 5000-5009. (7) Lee, C. W. B.; Waugh, J. S.; Griffin, R. G. Solid-State NMR-Study of trehalose/ 1,2-dipalmitoyl-sn-phosphatidylcholine interactions. Biochemistry 1986, 25 (13), 3737- 3742. (8) Koster, K. L.; Webb, M. S.; Bryant, G.; Lynch, D. V. Interactions between soluble sugars and POPC (1-palmitoyl-2-oleoylphosphatidylcholine) during dehy- dration: vitrification of sugars alters the phase behavior of the phospholipid. Biochim. Biophys. Acta 1994, 1193, 143-150. (9) Wolfe, J.; Bryant, G. Freezing, drying, and/or vitrification of membrane- solute-water systems. Cryobiology 1999, 39, 103-129. (10) Koster, K. L.; Maddocks, K. J.; Bryant, G. Exclusion of maltodextrins from phosphatidylcholine multilayers during dehydration: effects on membrane- phase behaviour. Eur. Biophys. J. 2003, 32, 96-105. (11) Lenn e, T.; Bryant, G.; Garvey, C. J.; Keiderling, U.; Koster, K. L. Location of sugars in multilamellar membranes at low hydration. Physica B 2006, 385-386, 862-864. (12) Arakawa, T.; Timasheff, S. N. 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