Structure, function, and membrane integration of defensins.

Defensins comprise a structural class of small cationic peptides that exert broad-spectrum antimicrobial activities through membrane permeabilization. Their predominantly beta-sheet structure, stabilized by three disulfide bonds, distinguishes them from other antimicrobial peptides which typically form amphiphilic helices. Defensins bind to membranes electrostatically and subsequently form apparently multimeric pores. Recent structural and biophysical studies are beginning to provide insights into the process of permeabilization.

Eradication of interleukin 5-transfected J558L plasmacytomas in mice by hydrogen peroxide-generating Stealth liposomes.

Certain human tumors are extensively infiltrated by eosinophils and contain extracellular deposits of eosinophil peroxidase, which uses hydrogen peroxide as a substrate to produce highly toxic hypohalous acids. We hypothesized that J558L HI, an interleukin 5-transfected murine plasmacytoma that is infiltrated by numerous degranulating eosinophils, would be especially sensitive to killing by hydrogen peroxide generated by glucose oxidase (beta-D-glucose:oxygen-oxido reductase; EC 1.13.4). Here we report that 4 i.v. injections of 0.5 ml of hydrogen peroxide-generating, anionic Stealth liposomes containing 50 micrograms of glucose oxidase eradicated s.c. implants of 10(6) J558L HI plasmacytoma cells in 6 of 13 mice. By contrast, the J558L HI tumors grew rapidly in 13 of 13 untreated mice and in 10 of 10 mice treated with daily i.v. injections of 50 micrograms of unencapsulated (free) glucose oxidase (P = 0.002 by log-rank test of survival curves constructed using the Kaplan-Meier method). Antisense transfected J558L tumors that did not contain eosinophils were not eradicated by the peroxide-generating liposomes in any of the 10 mice that were tested. Treatment with the liposomes was well tolerated for the first three doses (given on days 3, 4, and 5 after tumor inoculation). The fourth dose given on day 10 produced significant allergic toxicity and was, therefore, omitted in a second trial with only minimal reduction in the therapeutic response. We conclude that peroxide-generating, anionic Stealth liposomes can eradicate plasmacytomas infiltrated by eosinophils in mice. Our results, therefore, suggest that peroxide-generating compounds may be a useful experimental approach for treating those human tumors that are naturally infiltrated by eosinophils but resistant to conventional therapies.

Hydrophobic interactions of peptides with membrane interfaces.

The thermodynamic principles underlying the structural stability of membrane proteins are difficult to obtain directly from whole proteins because of intractable problems related to insolubility in the aqueous phase and extreme stability in the membrane phase. The principles must therefore be surmised from studies of the interactions of small peptides with lipid bilayers. This review is concerned with the hydrophobic interactions of such peptides with the interfacial regions of lipid bilayers. We first develop a general framework for thinking about the thermodynamics of membrane protein stability that centers on interfacial interactions and review the structural and chemical evidence that supports this interface-centered point of view. We then describe an experimentally determined whole-residue interfacial hydrophobicity scale that reveals the central role of the peptide bond in partitioning and folding. Finally, we consider the complexity and diversity of interfacial interactions revealed by differences between side-chain hydrophobicities determined using different classes of peptides.

Folding of beta-sheets in membranes: specificity and promiscuity in peptide model systems.

The interactions that drive the folding of beta-barrel membrane proteins have not been well studied because there have been few available model systems for membrane beta-sheets. In this work, we expand on a recently described model system to explore the contributions of interstrand hydrogen bonds, side-chain/side-chain interactions and side-chain/membrane interactions to beta-sheet formation in membranes. These experiments are based on the observation that the hydrophobic hexapeptide acetyl-Trp-Leu-Leu-Leu-Leu-Leu-OH (AcWLLLLL) folds, cooperatively and reversibly, into oligomeric, antiparallel beta-sheets in phosphatidylcholine membranes. To systematically characterize the important interactions that drive beta-sheet formation in membranes, we have used circular dichroism spectroscopy to determine the membrane secondary structure of each member of a complete host-guest family of related peptides of the form AcWLL-X-LL, where X is one of the natural amino acids. Peptides with hydrophobic X-residues of any size or character (X=Ala, Val, Ile, Leu, Cys, Met, Phe and Trp) form similar beta-sheets in membranes, while peptides with any polar X-residue or Gly or Pro at the X-position are random-coils, even when bound to membranes at high concentrations. The observed membrane sheet preferences correlate poorly with intrinsic sheet propensity scales measured in soluble proteins, but they correlate well with several membrane hydrophobicity scales. These results support the idea that the predominant interactions of the side-chains in membrane-bound beta-sheets are with the membrane lipids, and that backbone hydrogen bonding is the major driving force for the stabilization of beta-sheets in membranes.

An amphipathic alpha-helix at a membrane interface: a structural study using a novel X-ray diffraction method.

The amphipathic alpha-helix is a recurrent feature of membrane-active proteins, peptides, and toxins. Despite extensive biophysical studies, the structural details of its affinity for membrane interfaces remain rather vague. We report here the first results of an effort to obtain detailed structural information about alpha-helices in membranes by means of a novel X-ray diffraction method. Specifically, we determined the transbilayer position and orientation of an archetypal class A amphipathic helical peptide in oriented fluid-state dioleoylphosphatidylcholine (DOPC) bilayers. The peptide, Ac-18A-NH2(Ac-DWLKAFYDKVAEKLKEAF-NH2), is a model for class A amphipathic helices of apolipoprotein A-I and other exchangeable lipoproteins. The diffraction method relies upon experimental determinations of absolute scattering-length density profiles along the bilayer normal and the transbilayer distribution of the DOPC double bonds by means of specific bromination, and molecular modeling of the perturbed lipid bilayer (derived using the transbilayer distribution of the double bonds) and the peptide. The diffraction results showed that Ac-18A-NH2was located in the bilayer interface and that its transbilayer distribution could be described by a Gaussian function with a 1/e-halfwidth of 4.5(+/-0.3) A located 17.1(+/-0.3) A from the bilayer center, close to the glycerol moiety. Molecular modeling suggested that Ac-18A-NH2is helical and oriented generally parallel with the bilayer plane. The helicity and orientation were confirmed by oriented circular dichroism measurements. The width of the Gaussian distribution, a measure of the diameter of the helix, indicated that the Ac-18A-NH2helix penetrated the hydrocarbon core to about the level of the DOPC double bonds. Bilayer perturbations caused by Ac-18A-NH2were surprisingly modest, consisting of a slight decrease in bilayer thickness with a concomitant shift of the double-bond distribution toward the bilayer center, as expected from a small increase in lipid-specific area caused by the peptide.

Folding of beta-sheet membrane proteins: a hydrophobic hexapeptide model.

Beta-sheets, in the form of the beta-barrel folding motif, are found in several constitutive membrane proteins (porins) and in several microbial toxins that assemble on membranes to form oligomeric transmembrane channels. We report here a first step towards understanding the principles of beta-sheet formation in membranes. In particular, we describe the properties of a simple hydrophobic hexapeptide, acetyl-Trp-Leu5 (AcWL5), that assembles cooperatively into beta-sheet aggregates upon partitioning into lipid bilayer membranes from the aqueous phase where the peptide is strictly monomeric and random coil. The aggregates, containing 10 to 20 monomers, undergo a relatively sharp and reversible thermal unfolding at approximately 60 degreesC. No pores are formed by the aggregates, but they do induce graded leakage of vesicle contents at very high peptide to lipid ratios. Because beta-sheet structure is not observed when the peptide is dissolved in n-octanol, trifluoroethanol or sodium dodecyl sulfate micelles, aggregation into beta-sheets appears to be an exclusive property of the peptide in the bilayer membrane interface. This is an expected consequence of the hypothesis that a reduction in the free energy of partitioning of peptide bonds caused by hydrogen bonding drives secondary structure formation in membrane interfaces. But, other features of interfacial partitioning, such as side-chain interactions and reduction of dimensionality, must also contribute. We estimate from our partitioning data that the free energy reduction per residue for aggregation is about 0.5 kcal mol-1. Although modest, its aggregate effect on the free energy of assembling beta-sheet proteins can be huge. This surprising finding, that a simple hydrophobic hexapeptide readily assembles into oligomeric beta-sheets in membranes, reveals the potent ability of membranes to promote secondary structure in peptides, and shows that the formation of beta-sheets in membranes is more facile than expected. Furthermore, it provides a basis for understanding the observation that membranes promote self-association of beta-amyloid peptides. AcWL5 and related peptides thus provide a good starting point for designing peptide models for exploring the principles of beta-sheet formation in membranes.

Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores.

Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 microM POPG) readily aggregated at HNP-2 concentrations above 1 microM, but no mixing of vesicle contents could be detected for concentrations as high as 2 microM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 microM POPG vesicles, 50% of entrapped solute is released by 0.7 microM HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (approximately 400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.

Visual detection of specific, native interactions between soluble and microbead-tethered alpha-helices from membrane proteins.

Using peptides tethered to polymer microbeads, we have developed a technique for measuring the interactions between the transmembrane alpha-helices of membrane proteins and for screening combinatorial libraries of peptides for members that interact with specific helices from membrane proteins. The method was developed using the well-characterized homodimerization sequence of the membrane-spanning alpha-helix from the erythrocyte membrane protein glycophorin A (GPA). As a control, we also tested a variant with a dimer-disrupting alteration of a critical glycine residue to leucine. To test for detectable, native interactions between detergent-solubilized and microbead-tethered alpha-helices, we incubated fluorescent dye-labeled GPA analogues in sodium dodecyl sulfate solution with microbeads that contained covalently attached GPA analogues. When the dye-labeled peptide in solution and the bead-tethered peptide both contained the native glycophorin A sequence, the microbeads readily accumulated the dye through lateral peptide-peptide interactions and were visibly fluorescent under UV light. When either the peptide in solution or the peptide attached to the beads contained the glycine to leucine change, the beads did not accumulate any dye. The usefulness of this method for screening tethered peptide libraries was tested by incubating dye-labeled, native sequence peptides in detergent solution with a few native sequence beads plus an excess of beads containing the variant glycine to leucine sequence. When the dye-labeled peptide in solution was present at a concentration of > or =2 microM, the few native sequence beads were visually distinguishable from the others because of their bright fluorescence. Using this model system, we have shown that it is possible to visually detect specific, native interactions between alpha-helices from membrane proteins using peptides tethered to polymer microbeads. It will thus be possible to use this method to measure the specific lateral interactions that drive the folding and organization of membrane proteins and to screen combinatorial libraries of peptides for members that interact with them.

Quantitation of electrostatic and hydrophobic membrane interactions by equilibrium dialysis and reverse-phase HPLC.

Equilibrium dialysis and reverse-phase HPLC have been used for the sensitive and precise quantitation of both electrostatic and hydrophobic interactions of peptides and small molecules with lipid bilayers. We show that hydrophobic solutes are rapidly and quantitatively released from lipid dispersions when loaded onto a C4 reverse-phase HPLC column equilibrated in water+0.1% trifluoroacetic acid and that the lipid molecules have no interfering effect on the chromatography. Peptides interacting electrostatically with bilayers are released quantitatively when a higher ionic strength buffer (water+2% ammonium acetate) is used. As little as 50 ng of solute can be accurately quantitated even in the presence of milligram amounts of lipid. We demonstrate the application of these methods to the hydrophobic interactions between indoles and lipid bilayers and to the electrostatic interaction between defensins, which are cationic antibiotic peptides, and anionic bilayers. The high sensitivity allows nondestructive quantitation of submicrogram amounts of precious solutes and the high precision allows the heat capacity change, an important thermodynamic parameter, to be obtained from the partitioning data.

Exchange and flip-flop of dimyristoylphosphatidylcholine in liquid-crystalline, gel, and two-component, two-phase large unilamellar vesicles.

The rate and extent of spontaneous exchange of dimyristoylphosphatidylcholine (DMPC) from large unilamellar vesicles (LUV) composed of either DMPC or mixtures of DMPC/distearoylphosphatidylcholine (DSPC) have been examined under equilibrium conditions. The phase state of the vesicles ranged from all-liquid-crystalline through mixed gel/liquid-crystalline to all-gel. The exchange rate of DMPC between liquid-crystalline DMPC LUV, measured between 25 and 55 degrees C, was found to have an Arrhenius activation energy of 24.9 +/- 1.4 kcal/mol. This activation energy and the exchange rates are very similar to those obtained for the exchange of DMPC between DMPC small unilamellar vesicles (SUV). The extent of exchange of DMPC in LUV was found to be approximately 90%. This is in direct contrast to the situation in DMPC SUV where only the lipid in the outer monolayer is available for exchange. Thus, transbilayer movement (flip-flop) is substantially faster in liquid-crystalline DMPC LUV than in SUV. Desorption from gel-phase LUV has a much lower rate than gel-phase SUV with an activation energy of 31.7 +/- 3.7 kcal/mol compared to 11.5 +/- 2 kcal/mol reported for SUV. A defect-mediated exchange in gel-phase SUV, which is not the major pathway for exchange in LUV, is proposed on the basis of the thermodynamic parameters of the activation process. Surprisingly, the rates of DMPC exchange between DMPC/DSPC two-component LUV, measured over a wide range of compositions and temperatures, were found to exhibit very little dependence on the composition or phase configuration of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Transbilayer and interbilayer phospholipid exchange in dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine large unilamellar vesicles.

The rates of spontaneous interbilayer and transbilayer exchange of [3H]dimyristoylphosphatidylcholine ([3H]DMPC) were examined in DMPC and DMPC/dimyristoylphosphatidylethanolamine (DMPE) large unilamellar vesicles in the liquid-crystalline-, gel-, and mixed-phase states. DMPC desorption rates from either gel or liquid-crystalline phases containing DMPE are very similar to the corresponding rates from pure DMPC gel or liquid-crystalline phases. This is not the case for DMPC desorption from distearoylphosphatidylcholine (DSPC)-containing gel phases, where the desorption rates are significantly faster than from a pure DMPC gel phase [Wimley, W. C., & Thompson, T. E. (1990) Biochemistry 29, 1296-1303]. We proposed that the DMPC/DSPC behavior results from packing defects in gel phases composed of both DMPC and DSPC molecules because of the four-carbon difference in the acyl chain lengths of the two species. The present results strongly support this hypothesis because no such anomalous behavior is observed in DMPC/DMPE, which is similar to DMPC/DSPC in phase behavior but does not have the chain length difference. The inclusion of 10-30 mol % DMPE in DMPC bilayers was also found to have a significant effect on the rate of transbilayer movement (flip-flop) of [3H]DMPC in the liquid-crystalline phase. Between 10 and 30 mol % DMPE, flip-flop of DMPC is slowed by at least 10-fold relative to flip-flop in DMPC bilayers, and the entropy and enthalpy of flip-flop activation are both substantially decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphatidylethanolamine enhances the concentration-dependent exchange of phospholipids between bilayers.

It has previously been demonstrated that lipid exchange between phosphatidylcholine vesicles, at higher concentrations, is characterized by a second-order concentration-dependent exchange process in addition to the first-order process operative at lower concentrations (Jones, J. D., & Thompson, T. E. (1989) Biochemistry 28, 129-134). Furthermore, it was demonstrated that the second-order process occurs as a result of an enhancement of the first-order desorption process, possibly resulting from attractive interactions between a potentially desorbing lipid molecule and a transiently apposed bilayer (Jones, J. D., & Thompson, T. E. (1990) Biochemistry 29, 1593-1600). In this work we have studied the exchange of [3H]dimyristoylphosphatidylcholine (DMPC) between large vesicles of the compositions 100% DMPC, 70/30 (mol/mol) DMPC/dimyristoylphosphatidylethanolamine (DMPE), and 68.25/30/1.75 (mol/mol/mol) DMPC/DMPE/dimyristoylphosphatidylglycerol (DMPG). The second-order exchange process is enhanced by 100-fold or more in vesicles containing 30 mol % DMPE relative to 100% DMPC and is reduced or eliminated by the addition of 1.75% of the anionic lipid DMPG. These effects can be achieved by alterations in the equilibrium bilayer separation of 5 A or less. The results are in accord with the model of Jones and Thompson and indicate that relatively low concentrations of PE in a PC bilayer can have significant effects on bilayer surface properties and on potential interactions between bilayers.

Partitioning of tryptophan side-chain analogs between water and cyclohexane.

We have measured the partitioning of the tryptophan side-chain analogs 3-methylindole and N-methylindole between water and cyclohexane over the temperature range 8-55 degrees C to investigate the relative contribution of the imine-NH- to the free energy of transfer. We take advantage of the fact that the indole imine nitrogen is blocked by a methyl group in N-methylindole. Unlike previous studies, we take into account the water present in the cyclohexane phase. Free energies of partitioning were calculated using mole-fraction, volume-fraction, and Flory-Huggins-corrected volume-fraction partition coefficients [De Young, L. R., & Dill, K. A. (1990) J. Phys. Chem. 94, 801-809; Sharp, K. A., Nicholls, A., Friedman, R., & Honig, B. (1991) Biochemistry 30, 9686-9697]. These approaches account for configurational entropy changes in different ways and thus lead to different values for the calculated free energies of transfer. There is a 2-3-fold difference in the free energies calculated from our measurements, using the different units. Independent of units, the partitioning of both compounds involves identical entropy changes. However, 3-methylindole has an additional unfavorable enthalpic contribution to partitioning into cyclohexane of +1.6 kcal/mol (independent of units) which is presumably the cost of removing the indole -NH- group from water and transferring it to cyclohexane. In cyclohexane, 3-methylindole forms hydrogen bonds with water that cause water to copartition into cyclohexane with the solute. A method is described which allows the partitioning process to be examined independent of subsequent interactions with water in the solvent.

Designing transmembrane alpha-helices that insert spontaneously.

Direct measurement of the free energies of transfer of hydrophobic membrane-spanning alpha-helices from water to membranes is important for the determination of an accurate experiment-based hydrophobicity scale for membrane proteins. An important objective of such a scale is to account for the presently unknown thermodynamic cost of partitioning hydrogen-bonded peptide bonds into the membrane hydrocarbon core. We describe here the physical properties of a transmembrane (TM) peptide, TMX-1, designed to test the feasibility of engineering peptides that spontaneously insert across bilayers but that have the important property of measurable monomeric water solubility. TMX-1, Ac-WNALAAVAAAL-AAVAAALAAVAAGKSKSKS-NH(2), is a 31-residue sequence with a 21-residue nonpolar core, N- and C-caps to favor helix formation, and a highly polar C-terminus to improve solubility and to control directionality of insertion into lipid vesicles. TMX-1 appeared to be soluble in water up to a concentration of at least 1 mg/mL (0.3 mM). However, fluorescence spectroscopy, fluorescence quenching, and circular dichroism (CD) spectroscopy indicated that the high solubility was due to the formation of molecular aggregates that persisted at peptide concentrations down to at least 0.1 microM peptide. Nevertheless, aqueous TMX-1 partitioned strongly into membrane vesicles with apparent mole-fraction free-energy values of -7.1 kcal mol(-1) for phosphatidylcholine (POPC) vesicles and -8.2 kcal mol(-1) for phosphatidylglycerol (POPG) vesicles. CD spectroscopy of TMX-1 in oriented multilayers formed from either lipid disclosed a very strong preference for a transmembrane alpha-helical conformation. When TMX-1 was added to preformed vesicles, it was fully helical. A novel fluorescence resonance energy transfer (FRET) method demonstrated that at least 50% of the TMX-1 insered spontaneously across the vesicle membranes. Binding and insertion were found to be fully reversible for POPC vesicles but not POPG vesicles. TMX-1 was thus found to have many of the properties required for thermodynamic measurements of TM peptide insertion. Importantly, the results obtained delineate the experimental problems that must be considered in the design of peptides that can partition spontaneously and reversibly as monomers into and across membranes. Our success with TMX-1 suggests that these problems are not insurmountable.

Membrane protein folding and stability: physical principles.

Stably folded membrane proteins reside in a free energy minimum determined by the interactions of the peptide chains with each other, the lipid bilayer hydrocarbon core, the bilayer interface, and with water. The prediction of three-dimensional structure from sequence requires a detailed understanding of these interactions. Progress toward this objective is summarized in this review by means of a thermodynamic framework for describing membrane protein folding and stability. The framework includes a coherent thermodynamic formalism for determining and describing the energetics of peptide-bilayer interactions and a review of the properties of the environment of membrane proteins--the bilayer milieu. Using a four-step thermodynamic cycle as a guide, advances in three main aspects of membrane protein folding energetics are discussed: protein binding and folding in bilayer interfaces, transmembrane helix insertion, and helix-helix interactions. The concepts of membrane protein stability that emerge provide insights to fundamental issues of protein folding.

Experimentally determined hydrophobicity scale for proteins at membrane interfaces.

The partitioning of membrane-active oligopeptides into membrane interfaces promotes the formation of secondary structure. A quantitative description of the coupling of structure formation to partitioning, which may provide a basis for understanding membrane protein folding and insertion, requires an appropriate free energy scale for partitioning. A complete interfacial hydrophobicity scale that includes the contribution of the peptide bond was therefore determined from the partitioning of two series of small model peptides into the interfaces of neutral (zwitterionic) phospholipid membranes. Aromatic residues are found to be especially favoured at the interface while charged residues, and the peptide bond, are disfavoured about equally. Reduction of the high cost of partitioning the peptide bond through hydrogen bonding may be important in the promotion of structure formation in the membrane interface.

A high-throughput screen for identifying transmembrane pore-forming peptides.

We have developed a visual microwell plate assay for rapid, high-throughput screening for membrane-disrupting molecules such as de novo designed pore formers, antibiotic peptides, bacterial toxins, and lipases. The detectability is based on the strong fluorescence emission of the lanthanide metal terbium(III) (Tb(3+)) when it interacts with the aromatic chelator dipicolinic acid (DPA). While Tb(3+) is not strongly fluorescent alone, the binary complex emits bright green fluorescence when irradiated with uv light. For the microwell plate assay, we prepared unilamellar phospholipid vesicles that had either Tb(3+) or DPA entrapped and the opposite molecule in the external solution. Disruption of the membranes allows the Tb(3+)/DPA complex to form, giving rise to a visibly fluorescent solution. In plates with 20-microl wells, the lower limit of visual detectability of the Tb(3+)/DPA complex in solution was about 2.5 microM. The lower limit of detectability using vesicles with entrapped Tb(3+) or DPA was about 50 microM phospholipid. We show that the membrane-disrupting effect of as little as 0.25 microM or 5 pmol of the pore-forming, antibiotic peptide alamethicin can be detected visually with this system. This sensitive, high-throughput assay is readily automatable and makes possible the visual screening of combinatorial peptide libraries for members that permeabilize lipid bilayer membranes.

Membrane partitioning: distinguishing bilayer effects from the hydrophobic effect.

The free energy of transfer of nonpolar solutes from water to lipid bilayers is often dominated by a large negative enthalpy rather than the large positive entropy expected from the hydrophobic effect. This common observation has led to the concept of the "nonclassical" hydrophobic effect and the idea that the "classical" hydrophobic effect may not drive partitioning in many bilayer systems. We show through measurements of the heat capacity changes associated with the partitioning of tryptophan side-chain analogs into lipid bilayers and into bulk cyclohexane that the hydrophobic effect plays a crucial role regardless of the large negative enthalpy. The results emphasize that bulk-phase measurements are inadequate for describing bilayer partitioning. The experimental approach described should be generally useful for analyzing the bilayer interactions of a broad range of biologically important molecules.