Phase Behavior of a Phospholipid/Fatty Acid/Water Mixture Studied in Atomic Detail Volker Knecht,* ,² Alan E. Mark, ‡,§ and Siewert-Jan Marrink § Contribution from the Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany, School of Molecular and Microbial Science, UniVersity of Queensland, St Lucia, Brisbane QLD 4072, Australia, and Department of Biophysical Chemistry, UniVersity of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Received September 27, 2005; E-mail: vknecht@mpikg.mpg.de Abstract: Molecular dynamics simulations have been used to study the phase behavior of a dipalmi- toylphosphatidylcholine (DPPC)/palmitic acid (PA)/water 1:2:20 mixture in atomic detail. Starting from a random solution of DPPC and PA in water, the system adopts either a gel phase at temperatures below ∼330 K or an inverted hexagonal phase above ∼330 K in good agreement with experiment. It has also been possible to observe the direct transformation from a gel to an inverted hexagonal phase at elevated temperature (∼390 K). During this transformation, a metastable fluid lamellar intermediate is observed. Interlamellar connections or stalks form spontaneously on a nanosecond time scale and subsequently elongate, leading to the formation of an inverted hexagonal phase. This work opens the possibility of studying in detail how the formation of nonlamellar phases is affected by lipid composition and (fusion) peptides and, thus, is an important step toward understanding related biological processes, such as membrane fusion. Introduction During the biological processes of membrane fusion and budding, lipids must undergo a transition from lamellar to nonlamellar structures. 1 On the basis of predictions from continuum models, membrane fusion is believed to be initiated by the formation of interlamellar connections (stalks). 2,3 Stalks are also predicted as intermediates in the transformation from a lamellar to an inverted hexagonal phase. 4,5 Direct evidence for a stalk intermediate has come recently from the observation of a phase of stable stalks, the rhombohedral phase. 6 Whereas the phase behavior of lipid systems is routinely determined from experiment, the molecular details of phase transformations are difficult to assess. Computer simulations using simplified models have given a qualitative understanding of these processes. Although yielding conflicting results on the later stages of these processes, such studies support the hypothesis that the formation of stalks initiates the fusion of vesicles 7-11 and the transforma- tion from a lamellar to an inverted hexagonal phase. 12 Simplified models nevertheless have limitations. Although they make it possible to sample the time and length scales required to investigate phase transitions at a modest computa- tional cost, important details of the atomic interactions such as hydrogen bonds are lost. To go beyond a qualitative understand- ing and to verify the results obtained with simplified models, it is necessary to study the process of phase transformation in atomic or near atomic detail. The computational cost of such simulations has, however, meant that such studies have only recently become possible. In fact, to date, only one atomistic simulation of a transformation between two alternative non- lamellar lipid phases (from a cubic to an inverted hexagonal phase) has been published. 13 Unfortunately, in that work, the cubic phase was unstable under all conditions investigated, and thus, the transformation was not between two thermodynami- cally stable states. This meant that although the study shed much light on the transformation process, the results could not be directly related to experiment. To reliably simulate phase transformations, it is essential that the model used accurately reproduces the phase behavior of a lipid system. This is particularly challenging, as the phase of a lipid system depends on a subtle balance of forces between the lipid headgroups and tails. It also means that the ability to correctly reproduce phase behavior is a very stringent test of the validity of the atomic models used in simulations. To study if the lamellar/nonlamellar phase behavior of a lipid system can be reproduced using an atomistic model, we have performed a series of molecular dynamics simulations of a dipalmitoylphosphatidylcholine (DPPC)/palmitic acid (PA)/ ² Max Planck Institute of Colloids and Interfaces. ‡ University of Queensland. § University of Groningen. (1) Markin, V. S.; Kozlov, M. M.; Borovjagin, V. L. Gen. Physiol. Biophys. 1984, 3, 361-377. (2) Kozlov, M. M.; Markin, V. S. Biofizika 1983, 28, 242-247. (3) Chernomordik, L. V.; Melikyan, G. B.; Chizmadzhev, Y. A. Biochim. Biophys. Acta 1987, 906, 309-352. (4) Siegel, D. P.; Epand, R. M. Biophys. J. 1997, 73, 3089-3111. (5) Siegel, D. P. Biophys. J. 1999, 291-313, 1999. (6) Yang, L.; Huang, H. W. Science 2002, 297, 1877-1879. (7) Noguchi, H.; Takasu, M. J. Chem. Phys. 2001, 115, 9547-9551. (8) Marrink, S. J.; Mark, A. E. J. Am. Chem. Soc. 2003, 125, 11144-11145. (9) Mu ¨ller, M.; Katsov, K.; Schick, M. Biophys. J. 2003, 85, 1611-1623. (10) Stevens, M. J.; Hoh, J. H.; Woolf, T. B. Phys. ReV. Lett. 2003, 91, 188102. (11) Shillcock, J. C.; Lipowsky, R. Nat. Mater. 2005, 4, 225-228. (12) Marrink, S. J.; Mark, A. E. Biophys. J. 2004, 87, 3894-3900. (13) Marrink, S. J.; Tieleman, D. P. Biophys. J. 2002, 83, 2386-2392. Published on Web 01/19/2006 2030 9 J. AM. CHEM. SOC. 2006, 128, 2030-2034 10.1021/ja056619o CCC: $33.50 © 2006 American Chemical Society