Interactions of alpha-helices with lipid bilayers: a review of simulation studies

Membrane proteins, of which the majority seem to contain one or more alpha-helix, constitute approx. 30% of most genomes. A complete understanding of the nature of helix/bilayer interactions is necessary for an understanding of the structural principles underlying membrane proteins. This review describes computer simulation studies of helix/bilayer interactions. Key experimental studies of the interactions of alpha-helices and lipid bilayers are briefly reviewed. Surface associated helices are found in some membrane-bound enzymes (e.g. prostaglandin synthase), and as stages in the mechanisms of antimicrobial peptides and of pore-forming bacterial toxins. Transmembrane alpha-helices are found in most integral membrane proteins, and also in channels formed by amphipathic peptides or by bacterial toxins. Mean field simulations, in which the lipid bilayer is approximated as a hydrophobic continuum, have been used in studies of membrane-active peptides (e.g. alamethicin, melittin, magainin and dermaseptin) and of simple membrane proteins (e.g. phage Pf1 coat protein). All atom molecular dynamics simulations of fully solvated bilayers with transmembrane helices have been applied to: the constituent helices of bacteriorhodopsin; peptide-16 (a simple model TM helix); and a number of pore-lining helices from ion channels. Surface associated helices (e.g. melittin and dermaseptin) have been simulated, as have alpha-helical bundles such as bacteriorhodopsin and alamethicin. From comparison of the results from the two classes of simulation, it emerges that a major theoretical challenge is to exploit the results of all atom simulations in order to improve the mean field approach.     

Structural Polymorphism of Gramicidin A Channels: Ion Conductivity and Spectral Studies

The relation between the various spatial structures of the gramicidin A channels and their ionic conductance has been studied. For this aim, various conformations of the peptide were pre-formed in liposomal bilayer and after subsequent fusion of liposomes with planar lipid bilayer the measured channel conductance was correlated with gramicidin structures established in liposomes. To form the single-stranded π6.3π 6.3 helix the peptide and lipid were co-dissolved in TFE prior to liposome preparation. THF and other solvents were used to form parallel (↑ ↑ π π) and antiparallel (↑ ↓ π π) double helices. Conformation of gramicidin in liposomes made by various phosphatidylcholines was monitored by CD spectroscopy, and computer analysis of the spectra obtained was performed. After fusion of gramicidin containing liposomes with planar bilayer membranes from asolectin, the histograms of single-channel conductance were obtained. The histograms had one or three distinct peaks depending on the liposome preparation. Assignment of the structure of the channel to conductance levels was made by correlation of CD data with conductance histograms. The channel-forming analogue, des(Trp-Leu)₂-gramicidin A, has been studied by the same protocol. The channel conductances of gramicidin A and the shortened analogue increase in the following order: ↑ ↓ π π 2 ↑ ↑ π π < π 6.3π6.3. Single-channels formed by double helices have higher dispersity of conductance than the π6.3π6.3 helical channel. Lifetimes of the double helical and the π6.3π6.3 helical channels are very close to each other. The data obtained were compared with theoretically predicted properties of double helices [1].     

Orientational preferences of neighboring helices can drive ER insertion of a marginally hydrophobic transmembrane helix

α-helical integral membrane proteins critically depend on the correct insertion of their transmembrane α helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane α helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (N(cyt)-C(lum) or N(lum)-C(cyt)) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the "missing hydrophobicity" in polytopic membrane proteins.     

Essential role of the conformational flexibility of helices 1 and 5 on the lipid binding activity of apolipophorin-III.

It has been recently postulated that the conformational flexibility of helices 1 and 5 of Locusta migratoria apoLp-III could play an important role in early steps of binding of this apolipoprotein to a lipid surface (Soulages, J. L., and Arrese, E. L. (2000) J. Biol. Chem. 275, 17501-17509). To test this model, we have designed a double Cys mutant in which a disulfide bond linking helices 1 and 5 could be formed, resulting in an apolipoprotein with reduced conformational flexibility of its N- and C-terminal helices. Substitution of Thr(18) and Ala(147) by Cys residues provided a protein that under nonreducing conditions was fully oxidized. The far-UV CD spectra of this mutant in the reduced and oxidized states indicated that their secondary structures were identical to the structure of the wild type recombinant apoLp-III, which contains no Cys residues. Near-UV CD studies confirmed the formation of a disulfide bond and the absence of structural perturbations. The lipid binding activity of the reduced mutant, as determined by its ability to form discoidal lipoproteins, was nearly identical to that of the wild type protein. Contrarily, the disulfide form of the mutant was not able to form discoidal lipoproteins with liposomes of either dimirystoylphosphatidylcholine or dimyristoylphosphatidylglycerol. It is concluded that the separation of the helices 1 and 5 constitutes one of the key steps along the complex pathway for the formation of the final apolipoprotein lipid-bound state. It is inferred that the conformational flexibility of helices 1 and 5 is a key property of apoLp-III, allowing the exposure of hydrophobic protein regions and the interaction of the hydrophobic faces of the amphipathic alpha-helices with the lipoprotein lipid surface.     

Ion-channel formation assisted by electrostatic interhelical interactions in covalently dimerized amphiphilic helical peptides

An ultimate goal of synthetic ion-channel peptide design is to construct stable and functional ion-conducting pores. It is expected that specific interhelical interactions would facilitate the association of helices in phospholipid membranes and the successive helix-bundle formation. In the present study, we rationally designed helix-bundle ion channels using the synthetic hybrid peptide K20E20, a disulfide dimer of cationic- and anionic-amphiphilic helices Ac-CGG-(BKBA) 5-NH 2 and Ac-CGG-(BEBA) 5-NH 2. Circular dichroism (CD) measurements in aqueous media implied helix stabilization in the peptide caused by the interhelical electrostatic interactions. In addition, CD spectra recorded in the presence of DPPC liposomes and dye-leakage measurements suggested a high degree of association of peptide monomers in phospholipid membranes as well as high affinities between peptide and lipid bilayers. These features allowed ion-channel formation at extremely low peptide concentrations (as low as 1 nM). According to electrophysiological analyses, stable helix bundles were constructed of six peptide helices by association of three K20E20 molecules. Helix-helix association in lipid membranes, peptide-membrane interactions, and ion-channel formation of K20E20 peptides were all facilitated by intramolecular electrostatic interactions between the helices of the hybrid peptide and were pH-dependent. Conductance through K20E20 ion channels decreased under acidic conditions because of the interruption of the salt bridges.     

Exploring the role of the phospholipid ligand in endothelial protein C receptor: A molecular dynamics study

Endothelial protein C receptor (EPCR) is a CD1‐like transmembrane glycoprotein with important regulatory roles in protein C (PC) pathway, enhancing PC's anticoagulant, anti‐inflammatory, and antiapoptotic activities. Similarly to homologous CD1d, EPCR binds a phospholipid [phosphatidylethanolamine (PTY)] in a groove corresponding to the antigen‐presenting site, although it is not clear if lipid exchange can occur in EPCR as in CD1d. The presence of PTY seems essential for PC γ‐carboxyglutamic acid (Gla) domain binding. However, the lipid‐free form of the EPCR has not been characterized. We have investigated the structural role of PTY on EPCR, by multiple molecular dynamics (MD) simulations of ligand bound and unbound forms of the protein. Structural changes, subsequent to ligand removal, led to identification of two stable and folded ligand‐free conformations. Compared with the bound form, unbound structures showed a narrowing of the A′ pocket and a high flexibility of the helices around it, in agreement with CD1d simulation. Thus, a lipid exchange with a mechanism similar to CD1d is proposed. In addition, unbound conformations presented a reduced interaction surface for Gla domain, confirming the role of PTY in establishing the proper EPCR conformation for the interaction with its partner protein. Single MD simulations were also obtained for 29 mutant models with predicted structural stability and impaired binding ability. Ligand affinity calculations, based on linear interaction energy method, showed that substitution‐induced conformational changes affecting helices around the A′ pocket were associated to a reduced binding affinity. Mutants responsible for this effect may represent useful reagents for experimental tests. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Oriented circular dichroism of a class A amphipathic helix in aligned phospholipid multilayers

The effect of lipid phase state on the orientation and conformation of a class A alpha-helical peptide on aligned lipid multilayers was examined using oriented circular dichroism spectroscopy. A comparison of oriented spectra in aligned peptide-lipid multilayers with CD spectra of unaligned peptide lipid vesicle complexes is consistent with a preferential alignment of helices parallel to the membrane surface at temperatures above and below the main acyl-chain melting transition temperature of the phospholipid. Changes are observed in the oriented CD spectra with lipid phase state which are attributed to a subtle conformational change of the peptide on the lipid surface. The results are compared with available experimental data on membrane-active lytic and antimicrobial helical peptides.     

Conformation of membrane-associated proapoptotic tBid

The proapoptotic Bcl-2 family protein Bid is cleaved by caspase-8 to release the C-terminal fragment tBid, which translocates to the outer mitochondrial membrane and induces massive cytochrome c release and cell death. In this study, we have characterized the conformation of tBid in lipid membrane environments, using NMR and CD spectroscopy with lipid micelle and lipid bilayer samples. In micelles, tBid adopts a unique helical conformation, and the solution NMR (1)H/(15)N HSQC spectra have a single well resolved resonance for each of the protein amide sites. In lipid bilayers, tBid associates with the membrane with its helices parallel to the membrane surface and without trans-membrane helix insertion, and the solid-state NMR (1)H/(15)N polarization inversion with spin exchange at the magic angle spectrum has all of the amide resonances centered at (15)N chemical shift (70-90 ppm) and (1)H-(15)N dipolar coupling (0-5 kHz) frequencies associated with NH bonds parallel to the bilayer surface, with no intensity at frequencies associated with NH bonds in trans-membrane helices. Thus, the cytotoxic activity of tBid at mitochondria may be similar to that observed for antibiotic polypeptides, which bind to the surface of bacterial membranes as amphipathic helices and destabilize the bilayer structure, promoting the leakage of cell contents.     

Dynamics and hydration of the alpha-helices of apolipophorin III

Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein whose structure is represented as a bundle of five amphipathic alpha-helices. In order to study the properties of the helical domains of apolipophorin III, we designed and obtained five single-tryptophan mutants of Locusta migratoria apoLp-III. The proteins were studied by UV absorption spectroscopy, time-resolved and steady-state fluorescence spectroscopy, and circular dichroism. Fluorescence anisotropy, near-UV CD and solute fluorescence quenching studies indicate that the Trp residues in helices 1 (N-terminal) and 5 (C-terminal) have the highest conformational flexibility. These two residues also showed the highest degree of hydration. Trp residues in helices 3 and 4 display the lowest mobility, as assessed by fluorescence anisotropy and near UV CD. The Trp residue in helix 2 is protected from the solvent but shows high mobility. As inferred from the properties of the Trp residues, helices 1 and 5 appear to have the highest conformational flexibility. Helix 2 has an intermediate mobility, whereas helices 3 and 4 appear to constitute a highly ordered domain. From the configuration of the helices in the tertiary structure of the protein, we estimated the relative strength of the five interhelical interactions of apoLp-III. These interactions can be ordered according to their apparent stabilizing strengths as: helix 3-helix 4 > helix 2-helix 3 > helix 4-helix 1 approximately helix 2-helix 5 > helix 1-helix 5. A new model for the conformational change that is expected to occur upon binding of the apolipoprotein to lipid is proposed. This model is significantly different from the currently accepted model (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesemberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, M. (1991) Biochemistry 30, 603-608). The model presented here predicts that the relaxation of the tertiary structure and the concomitant exposure of the hydrophobic core take place through the disruption of the weak interhelical contacts between helices 1 and 5. To some extent, the weakness of the helix 1-helix 5 interaction would be due to the parallel arrangement of these helices.     

Cooperative effects in α-helices: An ab initio molecular-orbital study

Some properties of α-helices of polyclycine and polyalanine, up to the decapeptide, were investigated by ab initio molecular-orbital calculations. These helices were found to be unstable relative to the corresponding “fully extended chain” conformation. The electric field of helices of 8–10 residues is about 20% stronger than that of models built from noninteracting monomers, for example. This is a result of cooperativity, which is essentially governed by the intramolecular hydrogen bonds. The cooperativity is manifest in all properties of the helices: relative stability, dipole moment, proton affinity, electrical potential. The electric potential of helices of three and four residues is such that their instability can be compensated for by a single charged group acting as an “initiator.” The computed proton affinity of the (Ala)₈ α-helix is about 45 kcal/mol larger than that of formamide, which confirms that long helices may be protonated at the carboxyl end in solution.     

Signal transduction by CD58: The transmembrane isoform transmits signals outside lipid rafts independently of the GPI-anchored isoform

The adhesion molecule CD58 is natively expressed in both a glycosylphosphatidylinositol (GPI)-anchored form and a transmembrane form. We previously demonstrated that the two isoforms of CD58 are differentially distributed in the cell membrane. The GPI-linked form resides in lipid rafts while the transmembrane form resides outside lipid rafts. Following cross-linking a fraction of transmembrane CD58 redistributes to lipid rafts. It has also been demonstrated that ligand binding to CD58 induces biological functions such as cytokine production and immunoglobulin isotype switching, indicating that cell–cell interactions result in CD58-mediated signal transduction. However, the signaling pathways involved in these activation processes are poorly defined. Here we show for the first time that cross-linking of CD58 induces protein tyrosine phosphorylation of BLNK, Syk and PLCγ, and activation of ERK and Akt/PKB. In addition, we studied how these signaling events relate to the distinct membrane localization of the two isoforms of CD58. We demonstrate that cross-linking of CD58 triggers signaling that is predominantly associated with transmembrane CD58 in nonraft microdomains. Moreover, signaling through transmembrane CD58 does not depend on coexpression of the GPI-linked isoform. Thus, despite the residence of its GPI-anchored isoform in lipid rafts and the translocation of a fraction of its transmembrane isoform to lipid rafts, CD58 signaling is triggered by the transmembrane isoform outside lipid rafts. These findings corroborate signaling outside lipid rafts, as opposed to the established notion that rafts function as essential platforms for signaling.     

Molecular recognition of human CD1b antigen complexes: evidence for a common pattern of interaction with alpha beta TCRs

Ag-specific T cell recognition is mediated through direct interaction of clonotypic TCRs with complexes formed between Ag-presenting molecules and their bound ligands. Although characterized in substantial detail for class I and class II MHC encoded molecules, the molecular interactions responsible for TCR recognition of the CD1 lipid and glycolipid Ag-presenting molecules are not yet well understood. Using a panel of epitope-specific Abs and site-specific mutants of the CD1b molecule, we showed that TCR interactions occur on the membrane distal aspects of the CD1b molecule over the alpha1 and alpha2 domain helices. The location of residues on CD1b important for this interaction suggested that TCRs bind in a diagonal orientation relative to the longitudinal axes of the alpha helices. The data point to a model in which TCR interaction extends over the opening of the putative Ag-binding groove, making multiple direct contacts with both alpha helices and bound Ag. Although reminiscent of TCR interaction with MHC class I, our data also pointed to significant differences between the TCR interactions with CD1 and MHC encoded Ag-presenting molecules, indicating that Ag receptor binding must be modified to accommodate the unique molecular structure of the CD1b molecule and the unusual Ags it presents.     

Structure and dynamics of lipid-associated states of apocytochrome c.

Apocytochrome c (apocyt c), which in aqueous solution is largely unstructured, acquires an alpha-helical conformation upon association with lipid membranes. The extent of alpha-helix induced in apocyt c is lipid-dependent and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The structural and dynamic properties of apocyt c in lipid membranes were investigated by attenuated total reflection Fourier transform infrared spectroscopy combined with amide H-D exchange kinetics. Apocyt c acquires a higher content of alpha-helical structure with negatively charged membranes than with zwitterionic ones. For all membranes studied here, the helices of these partially folded states of apocyt c have a preferential orientation perpendicular to the plane of the lipid membrane. The H-D exchange revealed that a small fraction of amide protons of apocyt c, possibly associated with a stable folded domain protected by the lipid, remained protected from exchange over 20 min. However, a large fraction of amide protons exchanged in less than 20 min, indicating that the helical states of apocyt c in lipid membranes are very dynamic.     

Secondary structure of spiralin in solution, at the air/water interface, and in interaction with lipid monolayers

The surface of spiroplasmas, helically shaped pathogenic bacteria related to the mycoplasmas, is crowded with the membrane-anchored lipoprotein spiralin whose structure and function are unknown. In this work, the secondary structure of spiralin under the form of detergent-free micelles (average Stokes radius, 87.5 A) in water and at the air/water interface, alone or in interaction with lipid monolayers was analyzed. FT-IR and circular dichroism (CD) spectroscopic data indicate that spiralin in solution contains about 25+/-3% of helices and 38+/-2% of beta sheets. These measurements are consistent with a consensus predictive analysis of the protein sequence suggesting about 28% of helices, 32% of beta sheets and 40% of irregular structure. Brewster angle microscopy (BAM) revealed that, in water, the micelles slowly disaggregate to form a stable and homogeneous layer at the air/water interface, exhibiting a surface pressure up to 10 mN/m. Polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) spectra of interfacial spiralin display a complex amide I band characteristic of a mixture of beta sheets and alpha helices, and an intense amide II band. Spectral simulations indicate a flat orientation for the beta sheets and a vertical orientation for the alpha helices with respect to the interface. The combination of tensiometric and PMIRRAS measurements show that, when spiroplasma lipids are used to form a monolayer at the air/water interface, spiralin is adsorbed under this monolayer and its antiparallel beta sheets are mainly parallel to the polar-head layer of the lipids without deep perturbation of the fatty acid chains organization. Based upon these results, we propose a 'carpet model' for spiralin organization at the spiroplasma cell surface. In this model, spiralin molecules anchored into the outer leaflet of the lipid bilayer by their N-terminal lipid moiety are composed of two colinear domains (instead of a single globular domain) situated at the lipid/water interface. Owing to the very high amount of spiralin in the membrane, such carpets would cover most if not all the lipids present in the outer leaflet of the bilayer.     

A New Aspect in Glycolipid Biology: Glycosphingolipids as Antigens Recognized by T Lymphocytes

T cells may recognize a large variety of ligands with different chemical structures. Recently, glycosphingolipids have also been shown to stimulate human T lymphocytes. Recognition of glycosphingolipids is restricted by the nonpolymorphic CD1 molecules, expressed by professional antigen-presenting cells and by macrophages infiltrating inflammatory sites. CD1 molecules have a structure resembling that of classical MHC class I molecules, with the terminal extracellular domains characterized by two antiparallel a helices placed on two hydrophobic pockets. The glycosphingolipids bound to CD1 insert the lipid tails in the two pockets and position the hydrophilic head on the external part of CD1. The TCR interacts with aminoacids present in the two a helices and with residues provided by the carbohydrate moiety of glycosphingolipids and discriminates their structural variations. T cells recognizing self-glycosphingolipids release proinflammatory cytokines and may have a pathogenetic role in autoimmune diseases such as multiple sclerosis.     

Mechanically induced helix-coil transition in biopolymer networks

The quasi-equilibrium evolution of the helical fraction occurring in a biopolymer network (gelatin gel) under an applied stress has been investigated by observing modulation in its optical activity. Its variation with the imposed chain extension is distinctly nonmonotonic and corresponds to the transition of initially coiled strands to induced left-handed helices. The experimental results are in qualitative agreement with theoretical predictions of helices induced on chain extension. This new effect of mechanically stimulated helix-coil transition has been studied further as a function of the elastic properties of the polymer network: crosslink density and network aging.     

Conformation of human apolipoprotein C-I in a lipid-mimetic environment determined by CD and NMR spectroscopy.

The high-resolution conformation of human apoC-I in complexes with sodium dodecyl sulfate (SDS) is presented. As estimated from CD data, apoC-I adopts 54% helical secondary structure when bound to SDS, which is similar to the helical content previously found with phospholipids. The NMR-derived conformation of apoC-I is composed of two amphipathic helices, residues 7-29 and 38-52, separated by a flexible linker. The N-terminal helix contains a mobile hinge involving residues 12-15. The hydrophobic side chains cluster on the nonpolar face of both helices, thus forming two discrete lipid-binding sites in the N-terminal helix and one in the C-terminal helix. As suggested by amide proton resonance line widths and deuterium exchange rates, the N-terminal helix is more flexible and may bind less tightly to the detergent than the C-terminal helix. The different mobility of both helices appears to be related to side-chain composition, rather than length of the amphipathic helix, and may play a role in the function of apoC-I as an activator of lecithin:cholesterol acyltransferase (LCAT). A model is suggested in which the C-terminal helix serves as a lipid anchor while the N-terminal helix may hinge off the lipid surface to make specific contacts with LCAT.     

Stable interactions between the transmembrane domains of the adenosine A2A receptor

G-protein-coupled receptors (GPCRs) must properly insert and fold in the membrane to adopt a stable native structure and become biologically active. The interactions between transmembrane (TM) helices are believed to play a major role in these processes. Previous studies in our group showed that specific interactions between TM helices occur, leading to an increase in helical content, especially in weakly helical TM domains, suggesting that helix–helix interactions in addition to helix–lipid interactions facilitate helix formation. They also demonstrated that TM peptides interact in a similar fashion in micelles and lipid vesicles, as they exhibit relatively similar thermal stability and α-helicity inserted in SDS micelles to that observed in liposomes. In this study, we perform an analysis of pairwise interactions between peptides corresponding to the seven TM domains of the human A_2A receptor (A_2AR). We used a combination of Förster resonance energy transfer (FRET) measurement and circular dichroism (CD) spectroscopy to detect and analyze these interactions in detergent micelles. We found that strong and specific interactions occur in only seven of the 28 possible peptide pairs. Furthermore, not all interactions, identified by FRET, lead to a change in helicity. Our results identify stabilizing contacts that are likely related to the stability of the receptor and that are consistent with what is known about the three-dimensional structure and stability of rhodopsin and the β₂ adrenergic receptor.     

Fluorescence spectroscopy of single tryptophan mutants of apolipophorin-III in discoidal lipoproteins of dimyristoylphosphatidylcholine

The structure of the exchangeable apolipoprotein, apolipophorin-III from Locusta migratoria, apoLp-III, is described as a bundle of five amphipathic alpha-helices. To study the interaction of each of the helices of apoLp-III with a lipid surface, we designed five single-Trp mutants, each containing a Trp residue in a different alpha-helix. The Trp residues were located in the nonpolar domains of the amphipathic alpha-helices. The kinetics of the spontaneous interaction of the mutants with dimyristoylphosphatidylcholine (DMPC) indicated that all mutants behaved as typical exchangeable apolipoproteins. Circular dichroism in the far-UV indicated that all proteins have a high and similar helical content in the lipid-bound state. The interaction of the Trp residues with the lipid surface was investigated in recombinant lipoprotein particles made with DMPC. The properties of the Trp residues were investigated by fluorescence spectroscopy. These studies showed major changes in the spectroscopic properties of the Trp residues upon binding to lipid. These changes are observed with all single-Trp mutants, indicating that a major conformational change, which affects the properties of all helices, takes place upon binding to lipid. The position of the fluorescence maximum, the quenching efficiency of acrylamide as determined by steady-state and time-resolved fluorescence, and the fluorescence lifetimes of the single-Trp mutants suggest that helices 1, 4, and 5 interact with the nonpolar domains of the lipid. The properties of the Trp in helices 2 and 3 suggest that these helices adopt a different binding configuration than helices 1, 4, and 5. Helices 2 and 3 appear to be interacting with the polar headgroups of the phospholipids or constitute a different domain that does not interact with the lipid surface.     

Interaction of two intrinsically disordered plant stress proteins (COR15A and COR15B) with lipid membranes in the dry state

COR15A and COR15B form a tandem repeat of highly homologous genes in Arabidopsis thaliana. Both genes are highly cold induced and the encoded proteins belong to the Pfam LEA_4 group (group 3) of the late embryogenesis abundant (LEA) proteins. Both proteins were predicted to be intrinsically disordered in solution. Only COR15A has previously been characterized and it was shown to be localized in the soluble stroma fraction of chloroplasts. Ectopic expression of COR15A in Arabidopsis resulted in increased freezing tolerance of both chloroplasts after freezing and thawing of intact leaves and of isolated protoplasts frozen and thawed in vitro. In the present study we have generated recombinant mature COR15A and COR15B for a comparative study of their structure and possible function as membrane protectants. CD spectroscopy showed that both proteins are predominantly unstructured in solution and mainly α-helical after drying. Both proteins showed similar effects on the thermotropic phase behavior of dry liposomes. A decrease in the gel to liquid-crystalline phase transition temperature depended on both the unsaturation of the fatty acyl chains and lipid headgroup structure. FTIR spectroscopy indicated no strong interactions between the proteins and the lipid phosphate and carbonyl groups, but significant interactions with the galactose headgroup of the chloroplast lipid monogalactosyldiacylglycerol. These findings were rationalized by modeling the secondary structure of COR15A and COR15B. Helical wheel projection indicated the presence of amphipathic α-helices in both proteins. The helices lacked a clear separation of positive and negative charges on the hydrophilic face, but contained several hydroxylated amino acids.