Enantiospecific Interactions between Cholesterol and Phospholipids Juha-Matti Alakoskela,* ,² Karen Sabatini, ² Xin Jiang, ‡ Venla Laitala, ² Douglas F. Covey, ‡ and Paavo K. J. Kinnunen ²,§ Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry, P.O. Box 63, UniVersity of Helsinki, 00014 Helsinki, Finland, Department of Molecular Biology and Pharmacology, Washington UniVersity School of Medicine, St. Louis, Missouri 63110, and MEMPHYSsCenter for Biomembrane Physics, UniVersity of Southern Denmark, Odense, Denmark ReceiVed September 19, 2007. In Final Form: October 26, 2007 The effects of cholesterol on various membrane proteins have received considerable attention. An important question regarding each of these effects is whether the cholesterol exerts its influence by binding directly to membrane proteins or by changing the properties of lipid bilayers. Recently it was suggested that a difference in the effects of natural cholesterol and its enantiomer, ent-cholesterol, would originate from direct binding of cholesterol to a target protein. This strategy rests on the fact that ent-cholesterol has appeared to have effects on lipid films similar to those of cholesterol, yet fluorescence microscopy studies of phospholipid monolayers have provided striking demonstrations of the enantiomer effects, showing opposite chirality of domain shapes for phospholipid enantiomer pairs. We observed the shapes of ordered domains in phospholipid monolayers containing either cholesterol or ent-cholesterol and found that the phospholipid chirality had a great effect on the domain chirality, whereas a minor (quantitative) effect of cholesterol chirality could be observed only in monolayers with racemic dipalmitoylphosphatidylcholine. The latter is likely to derive from cholesterol-cholesterol interactions. Accordingly, cholesterol chirality has only a modest effect that is highly likely to require the presence of solidlike domains and, accordingly, is unlikely to play a role in biological membranes. Introduction The demonstrations of enantiomer specificity in interactions between phospholipids and other small molecules are rare, with only a few reports in the literature, 1-3 while most studies of enantiomer interactions with phospholipids have failed to demonstrate any difference between enantiomers. 4-6 In contrast, enantiomer specificity is very frequent in interactions of compounds with proteins, since proteins typically have shaped surfaces devoid of symmetry and have multiple different interaction sites. The modulation of the functions of membrane proteins by lipids, lipophilic drugs, or other compounds interacting strongly with membranes always raises the question of whether this modulation derives from the direct binding of the lipid or drug onto membrane proteins or from the modulation of the physical properties of the membranes by these compounds. There are numerous ways in which the physical properties of membranes affect or could affect protein function (see, e.g., refs 7-11), and on the other hand, on the basis of lipid mobility studies and the increasing number of high-resolution structures of integral membrane proteins, these proteins are known to have several specific binding sites for different lipid species. 12,13 One good way to separate specific effects from the nonspecific ones could be to use enantiomers of lipid or drug molecules: if the effects of these enantiomers on lipids themselves are equal, then any demonstration of enantiospecificity in the effects on proteins provides proof of binding to proteins. This strategy has been outlined in the review by Westover and Covey, 14 suggesting that the enantiomer of cholesterol is an excellent tool to determine whether the effects of cholesterol on protein function derive from changes in the physical properties or from the binding of cholesterol on proteins. This suggestion, of course, relies on the fact that ent-cholesterol in phospholipid films has appeared to always produce equal effects compared to those of cholesterol. 14 Yet, while no difference between ent-cholesterol and cholesterol has been observed so far, the most definitive test to demonstrate the lack or presence of the enantiospecific interactions would appear to be to compare ent-cholesterol and cholesterol under conditions where phospholipids themselves have been shown to have enantiospecific effects or where cholesterol chirality has been suggested to have some significance. If some conditions exist where lipid films devoid of proteins display enantioselec- tivity, then in such conditions the interpretation of the enanti- oselectivity as direct protein binding should be taken with care. A striking demonstration of enantiomer effects comes from fluorescence microscopy of dipalmitoylphosphatidylcholine (DPPC) monolayers, where the large solidlike domains display opposite chirality for (R)-DPPC (also known as L-DPPC) and (S)-DPPC (also known as D-DPPC). 15 In the presence of small amounts of cholesterol they form spiral-shaped solid domains * To whom correspondence should be addressed. Phone: +358 9 19125426. Fax: +358 9 1912544. E-mail: jmalakos@cc.helsinki.fi. ² University of Helsinki. ‡ Washington University School of Medicine. § University of Southern Denmark. (1) Abood, L. G.; Hoss, W. P. Psychopharmacol. Commun. 1975, 1, 29-35. (2) Pathirana, S.; Neely, W. C.; Myers, L.; Vodyanoy, V. J. Am. Chem. Soc. 1992, 114, 1404-1405. (3) Nandi, N. J. Phys. Chem. A 2003, 107, 4588-4591. (4) Dickinson, B.; Franks, N. P.; Lieb, W. R. Biophys. J. 1994, 66, 2019- 2023. (5) Tomlin, S. L.; Jenkins, A.; Lieb, W. R.; Franks, N. P. Anesthesiology 1998, 88, 708-717. (6) Alakoskela, J.-M.; Covey, D. F.; Kinnunen, P. K. J. Biochim. Biophys. Acta 2007, 1768, 131-145. (7) Mouritsen, O. G.; Bloom, M. Biophys. J. 1984, 46, 141-153. (8) Cantor, R. S. J. Phys. Chem. B 1997, 101, 1723-1725. (9) De Planque, M. R. R.; Killian, J. A. Mol. Membr. Biol. 2003, 20, 271-284. (10) Lee, A. G. Biochim. Biophys. Acta 2004, 1666, 62-87. (11) Alakoskela, J.-M. Interactions in Lipid-Water Interface Assessed by Fluorescence Spectroscopy. Academic Dissertation. University of Helsinki, Finland, 2006; http://urn.fi/URN:ISBN:952-10-2884-X. (12) Marsh, D.; Pa ´li, T. Biochim. Biophys. Acta 2004, 1666, 118-141. (13) Qin, L.; Hiser, C.; Mulichak, A.; Garavito, R. M.; Ferguson-Miller, S. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16117-16122. (14) Westover, E. J.; Covey, D. F. J. Membr. Biol. 2004, 202, 61-72. (15) Weis, R. M.; McConnell, H. M. Nature 1984, 310, 47-49. 830 Langmuir 2008, 24, 830-836 10.1021/la702909q CCC: $40.75 © 2008 American Chemical Society Published on Web 01/03/2008