Transmembrane Peptides Stabilize Inverted Cubic Phases in a Biphasic Length-Dependent Manner: Implications for Protein-Induced Membrane Fusion D. P. Siegel,* V. Cherezov, y D. V. Greathouse, z R. E. Koeppe II, z J. Antoinette Killian, § and M. Caffrey y{ ** *Givaudan Inc., Cincinnati, Ohio; y Department of Chemistry, The Ohio State University, Columbus, Ohio; z Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas; § Department of Biochemistry of Membranes, University of Utrecht, Utrecht, The Netherlands; { Biochemistry and Biophysics Programs, The Ohio State University, Columbus, Ohio; and **College of Science, University of Limerick, Limerick, Ireland ABSTRACT WALP peptides consist of repeating alanine-leucine sequences of different lengths, flanked with tryptophan ‘‘anchors’’ at each end. They form membrane-spanning a-helices in lipid membranes, and mimic protein transmembrane domains. WALP peptides of increasing length, from 19 to 31 amino acids, were incorporated into N-monomethylated dioleoylphospha- tidylethanolamine (DOPE-Me) at concentrations up to 0.5 mol % peptide. When pure DOPE-Me is heated slowly, the lamellar liquid crystalline (L a ) phase first forms an inverted cubic (Q II ) phase, and the inverted hexagonal (H II ) phase at higher temperatures. Using time-resolved x-ray diffraction and slow temperature scans (1.5°C/h), WALP peptides were shown to decrease the temperatures of Q II and H II phase formation (T Q and T H , respectively) as a function of peptide concentration. The shortest and longest peptides reduced T Q the most, whereas intermediate lengths had weaker effects. These findings are relevant to membrane fusion because the first step in the L a /Q II phase transition is believed to be the formation of fusion pores between pure lipid membranes. These results imply that physiologically relevant concentrations of these peptides could increase the susceptibility of biomembrane lipids to fusion through an effect on lipid phase behavior, and may explain one role of the membrane-spanning domains in the proteins that mediate membrane fusion. INTRODUCTION WALP peptides are hydrophobic, a-helical transmembrane peptides that incorporate into lipid bilayers (1–5). The helical axes of such peptides are nearly perpendicular to the plane of the membrane at room temperature (1,3,4,6–8) and the core of the helices, when membrane-embedded, is strongly protected from solvent deuterium exchange (9). Because of these properties, WALP peptides are models for the single helical membrane-spanning domains found in some integral membrane proteins (1,4). WALP peptides stabilize inverted phases in phospholipids in a peptide-length- and concentra- tion-dependent fashion. In previous studies, peptides whose lengths as rigid a-helices are short compared to the thickness of the lamellar phase bilayer of the host lipid were generally found to be effective in inducing nonlamellar phase forma- tion, with the concentration required depending on the nature of the lipids. For example, it was shown that high concen- trations of short WALP peptides (;10 mol %) can induce formation of H II and so-called isotropic phases in fully hydrated phosphatidylcholine systems (1–3), which only form the lamellar liquid crystalline (L a ) phase in the absence of the peptides. Peptide concentrations of 1–4 mol % can substantially lower the L a /H II transition temperature and induce formation of an inverted cubic (Q II ) phase in 1,2- dielaidoyl-sn-3-glycero-3-phosphoethanolamine (DEPE) (10). WALP concentrations as small as 0.1 mol % substantially lowered the temperature for isotropic phase formation in a 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine/1,2- dioleoyl-sn-glycero-3-phosphoglycerol (DOPE/DOPG) mix- ture (11). Also, in previous studies, WALP peptides with lengths longer than the host lipid bilayer thickness were found to have little or no effect (10,11) on the phase behavior of the lipids. The fact that WALP peptides and other trans- membrane peptides (12) can stabilize nonlamellar (H II and Q II ) phases is especially interesting in light of the association of inverted phase behavior with membrane fusion (see, e.g., Ellens et al. (13) and Siegel (14)) and the possible influence of inverted phase behavior on membrane protein function (reviewed in Epand (15)). This work deals with the influence of WALP peptides on the formation of Q II and H II phases from the L a phase in DOPE-Me. These experiments were motivated by an interest in the physical chemical mechanisms by which proteins induce biomembrane fusion, which are still poorly un- derstood. There is a close relationship between Q II phase formation and the occurrence of membrane fusion in some pure lipid systems. Simply stated, formation of membrane fusion pores is postulated to be the first step in the L a /Q II phase transition (14,16). Membrane fusion rates increase substantially as unilamellar vesicle dispersions of DOPE-Me and DOPE/1,2-dioleoyl-sn-glycero-3-phosphocholine lipid mixtures are incubated at increasing temperatures in the region where Q II phase precursors and Q II phases are de- tected by 31 P NMR and freeze-fracture electron microscopy Submitted July 11, 2005, and accepted for publication September 13, 2005. Address reprint requests to David P. Siegel, Givaudan Inc., 1199 Edison Drive, Cincinnati, OH 45215. Tel.: 513-948-4840; E-mail: david.siegel@ givaudan.com. Ó 2006 by the Biophysical Society 0006-3495/06/01/200/12 $2.00 doi: 10.1529/biophysj.105.070466 200 Biophysical Journal Volume 90 January 2006 200–211