Host Defense Peptide Piscidin and Yeast-Derived Glycolipid Exhibit Synergistic Antimicrobial Action through Concerted Interactions with Membranes.

Developing new antimicrobials as alternatives to conventional antibiotics has become an urgent race to eradicate drug-resistant bacteria and to save human lives. Conventionally, antimicrobial molecules are studied independently even though they can be cosecreted in vivo. In this research, we investigate two classes of naturally derived antimicrobials: sophorolipid (SL) esters as modified yeast-derived glycolipid biosurfactants that feature high biocompatibility and low production cost; piscidins, which are host defense peptides (HDPs) from fish. While HDPs such as piscidins target the membrane of pathogens, and thus result in low incidence of resistance, SLs are not well understood on a mechanistic level. Here, we demonstrate that combining SL-hexyl ester (SL-HE) with subinhibitory concentration of piscidins 1 (P1) and 3 (P3) stimulates strong antimicrobial synergy, potentiating a promising therapeutic window. Permeabilization assays and biophysical studies employing circular dichroism, NMR, mass spectrometry, and X-ray diffraction are performed to investigate the mechanism underlying this powerful synergy. We reveal four key mechanistic features underlying the synergistic action: (1) P1/3 binds to SL-HE aggregates, becoming α-helical; (2) piscidin-glycolipid assemblies synergistically accumulate on membranes; (3) SL-HE used alone or bound to P1/3 associates with phospholipid bilayers where it induces defects; (4) piscidin-glycolipid complexes disrupt the bilayer structure more dramatically and differently than either compound alone, with phase separation occurring when both agents are present. Overall, dramatic enhancement in antimicrobial activity is associated with the use of two membrane-active agents, with the glycolipid playing the roles of prefolding the peptide, coordinating the delivery of both agents to bacterial surfaces, recruiting the peptide to the pathogenic membranes, and supporting membrane disruption by the peptide. Given that SLs are ubiquitously and safely used in consumer products, the SL/peptide formulation engineered and mechanistically characterized in this study could represent fertile ground to develop novel synergistic agents against drug-resistant bacteria.

Fungicidal amphotericin B sponges are assemblies of staggered asymmetric homodimers encasing large void volumes.

Amphotericin B (AmB) is a powerful but toxic fungicide that operates via enigmatic small molecule-small molecule interactions. This mechanism has challenged the frontiers of structural biology for half a century. We recently showed AmB primarily forms extramembranous aggregates that kill yeast by extracting ergosterol from membranes. Here, we report key structural features of these antifungal 'sponges' illuminated by high-resolution magic-angle spinning solid-state NMR, in concert with simulated annealing and molecular dynamics computations. The minimal unit of assembly is an asymmetric head-to-tail homodimer: one molecule adopts an all-trans C1-C13 motif, the other a C6-C7-gauche conformation. These homodimers are staggered in a clathrate-like lattice with large void volumes similar to the size of sterols. These results illuminate the atomistic interactions that underlie fungicidal assemblies of AmB and suggest this natural product may form biologically active clathrates that host sterol guests.

A Potent Host Defense Peptide Triggers DNA Damage and Is Active against Multidrug-Resistant Gram-Negative Pathogens.

Gram-negative bacteria are some of the biggest threats to public health due to a large prevalence of antibiotic resistance. The difficulty in treating bacterial infections, stemming from their double membrane structure combined with efflux pumps in the outer membrane, has resulted in a much greater need for antimicrobials with activity against these pathogens. Tunicate host defense peptide (HDP), Clavanin A, is capable of not only inhibiting Gram-negative growth but also potentiating activity in the presence of Zn(II). Here, we provide evidence that the improvements of Clavanin A activity in the presence of Zn(II) are due to its novel mechanism of action. We employed E. coli TD172 (ΔrecA::kan) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to show in cellulae that DNA damage occurs upon treatment with Clavanin A. In vitro assays demonstrated that Zn(II) ions are required for the nuclease activity of the peptide. The quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the mechanism of DNA damage. In the rate-determining step of the proposed mechanism, due to its Lewis acidity, the Zn(II) ion activates the scissile P-O bond of DNA and creates a hydroxyl nucleophile from a water molecule. A subsequent attack by this group to the electrophilic phosphorus cleaves the scissile phosphoester bond. Additionally, we utilized bacterial cytological profiling (BCP), circular dichroism (CD) spectroscopy in the presence of lipid vesicles, and surface plasmon resonance combined with electrical impedance spectroscopy in order to address the apparent discrepancies between our results and the previous studies regarding the mechanism of action of Clavanin A. Finally, our approach may lead to the identification of additional Clavanin A like HDPs and promote the development of antimicrobial peptide based therapeutics.

Enhancing the membrane activity of Piscidin 1 through peptide metallation and the presence of oxidized lipid species: Implications for the unification of host defense mechanisms at lipid membranes.

Piscidins are host-defense peptides (HDPs) from fish that exhibit antimicrobial, antiviral, anti-cancer, anti-inflammatory, and wound-healing properties. They are distinctively rich in histidine and contain an amino terminal copper and nickel (ATCUN) binding motif due to the presence of a conserved histidine at position 3. Metallation lowers their total charge and provides a redox center for the formation of radicals that can convert unsaturated fatty acids (UFAs) into membrane-destabilizing oxidized phospholipids (OxPLs). Here, we focus on P1, a particularly membrane-active isoform, and investigate how metallating it and making OxPL available influence its membrane activity. First, we quantify through dye leakage experiments the permeabilization of the apo- and holo-forms of P1 on model membranes containing a fixed ratio of anionic phosphatidylglycerol (PG) and zwitterionic phosphatidylcholine (PC) but varying amounts of Aldo-PC, an OxPL derived from the degradation of several UFAs. Remarkably, metallating P1 increases membranolysis by a factor of five in each lipid system. Conversely, making Aldo-PC available improves permeabilization by a factor of two for each peptide form. Second, we demonstrate through CD-monitored titrations that the strength of the peptide-membrane interactions is similar in PC/PG and PC/PG/Aldo-PC. Thus, peptide-induced membrane activity is boosted by properties intrinsic to the peptide (e.g., charge and structural changes associated with metallation) and bilayer (e.g., reversal of sn-2 chain due to oxidation). Third, we show using oriented-sample 15N solid-state NMR that the helical portion of P1 lies parallel to the bilayer surface in both lipid systems. 31P NMR experiments show that both the apo- and holo-states interact more readily with PC in PC/PG. However, the presence of Aldo-PC renders the holo-, but not the apo-state, more specific to PG. Hence, the membrane disruptive effects of P1 and its specificity for the anionic lipids found on pathogenic cell membrane surfaces are simultaneously optimized when it is metallated and the OxPL is present. Overall, this study deepens our insights into how OxPLs affect peptide-lipid interactions and how host defense metallopeptides could help integrate the effects of antimicrobial agents.

Lipopolysaccharide Simulations Are Sensitive to Phosphate Charge and Ion Parameterization.

The high proportion of lipopolysaccharide (LPS) molecules in the outer membrane of Gram-negative bacteria makes it a highly effective barrier to small molecules, antibiotic drugs, and other antimicrobial agents. Given this vital role in protecting bacteria from potentially hostile environments, simulations of LPS bilayers and outer membrane systems represent a critical tool for understanding the mechanisms of bacterial resistance and the development of new antibiotic compounds that circumvent these defenses. The basis of these simulations is parameterizations of LPS, which have been developed for all major molecular dynamics force fields. However, these parameterizations differ in both the protonation state of LPS and how LPS membranes behave in the presence of various ion species. To address these discrepancies and understand the effects of phosphate charge on bilayer properties, simulations were performed for multiple distinct LPS chemotypes with different ion parameterizations in both protonated or deprotonated lipid A states. These simulations show that bilayer properties, such as the area per lipid and inter-lipid hydrogen bonding, are highly influenced by the choice of phosphate group charges, cation type, and ion parameterization, with protonated LPS and monovalent cations with modified nonbonded parameters providing the best match to the experiments. Additionally, alchemical free energy simulations were performed to determine theoretical pKa values for LPS and subsequently validated by 31P solid-state nuclear magnetic resonance experiments. Results from these complementary computational and experimental studies demonstrate that the protonated state dominates at physiological pH, contrary to the deprotonated form modeled by many LPS force fields. Overall, these results highlight the sensitivity of LPS simulations to phosphate charge and ion parameters while offering recommendations for how existing models should be updated for consistency between force fields as well as to best match experiments.

Structure and Function in Antimicrobial Piscidins: Histidine Position, Directionality of Membrane Insertion, and pH-Dependent Permeabilization.

Piscidins are histidine-enriched antimicrobial peptides that interact with lipid bilayers as amphipathic α-helices. Their activity at acidic and basic pH in vivo makes them promising templates for biomedical applications. This study focuses on p1 and p3, both 22-residue-long piscidins with 68% sequence identity. They share three histidines (H3, H4, and H11), but p1, which is significantly more permeabilizing, has a fourth histidine (H17). This study investigates how variations in amphipathic character associated with histidines affect the permeabilization properties of p1 and p3. First, we show that the permeabilization ability of p3, but not p1, is strongly inhibited at pH 6.0 when the conserved histidines are partially charged and H17 is predominantly neutral. Second, our neutron diffraction measurements performed at low water content and neutral pH indicate that the average conformation of p1 is highly tilted, with its C-terminus extending into the opposite leaflet. In contrast, p3 is surface bound with its N-terminal end tilted toward the bilayer interior. The deeper membrane insertion of p1 correlates with its behavior at full hydration: an enhanced ability to tilt, bury its histidines and C-terminus, induce membrane thinning and defects, and alter membrane conductance and viscoelastic properties. Furthermore, its pH-resiliency relates to the neutral state favored by H17. Overall, these results provide mechanistic insights into how differences in the histidine content and amphipathicity of peptides can elicit different directionality of membrane insertion and pH-dependent permeabilization. This work features complementary methods, including dye leakage assays, NMR-monitored titrations, X-ray and neutron diffraction, oriented CD, molecular dynamics, electrochemical impedance spectroscopy, surface plasmon resonance, and quartz crystal microbalance with dissipation.

Calcium-Induced Lipid Nanocluster Structures: Sculpturing of the Plasma Membrane.

The plasma membrane of the cell is a complex, tightly regulated, heterogeneous environment shaped by proteins, lipids, and small molecules. Ca2+ ions are important cellular messengers, spatially separated from anionic lipids. After cell injury, disease, or apoptotic events, anionic lipids are externalized to the outer leaflet of the plasma membrane and encounter Ca2+, resulting in dramatic changes in the plasma membrane structure and initiation of signaling cascades. Despite the high chemical and biological significance, the structures of lipid-Ca2+ nanoclusters are still not known. Previously, we demonstrated by solid-state nuclear magnetic resonance (NMR) spectroscopy that upon binding to Ca2+, individual phosphatidylserine lipids populate two distinct yet-to-be-characterized structural environments. Here, we concurrently employ extensive all-atom molecular dynamics (MD) simulations with our accelerated membrane mimetic and detailed NMR measurements to identify lipid-Ca2+ nanocluster conformations. We find that major structural characteristics of these nanoclusters, including interlipid pair distances and chemical shifts, agree with observable NMR parameters. Simulations reveal that lipid-ion nanoclusters are shaped by two characteristic, long-lived lipid structures induced by divalent Ca2+. Using ab initio quantum mechanical calculations of chemical shifts on MD-captured lipid-ion complexes, we show that computationally observed conformations are validated by experimental NMR data. Both NMR measurements of diluted specifically labeled lipids and MD simulations reveal that the basic structural unit that reshapes the membrane is a Ca2+-coordinated phosphatidylserine tetramer. Our combined computational and experimental approach presented here can be applied to other complex systems in which charged membrane-active molecular agents leave structural signatures on lipids.

Peridinin Is an Exceptionally Potent and Membrane-Embedded Inhibitor of Bilayer Lipid Peroxidation.

Antilipoperoxidant protein dysfunction is associated with many human diseases, suggesting that bilayer lipid peroxidation may contribute broadly to pathogenesis. Small molecule inhibitors of this membrane-localized chemistry could in theory enable better understanding and/or treatment of such diseases, but currently available compounds have important limitations. Many biological questions thus remain unanswered, and clinical trials have largely been disappointing. Enabled by efficient, building block-based syntheses of three atypical carotenoid natural products produced by microorganisms that thrive in environments of extreme oxidative stress, we found that peridinin is a potent inhibitor of nonenzymatic bilayer lipid peroxidation in liposomes and in primary human endothelial cells. We also found that peridinin blocks monocyte-endothelial cell adhesion, a key step in atherogenesis. A series of frontier solid-state NMR experiments with a site-specifically 13C-labeled isotopolog synthesized using the same MIDA boronate building block-based total synthesis approach revealed that peridinin is completely embedded within and physically spans the hydrophobic core of POPC membranes, maximizing its effective molarity at the site of the targeted lipid peroxidation reactions. Alternatively, the widely used carotenoid astaxanthin is significantly less potent and was found to primarily localize extramembranously. Peridinin thus represents a promising and biophysically well-characterized starting point for the development of small molecule antilipoperoxidants that serve as more effective biological probes and/or therapeutics.

A Noncanonical Binding Site in the EVH1 Domain of Vasodilator-Stimulated Phosphoprotein Regulates Its Interactions with the Proline Rich Region of Zyxin.

Vasodilator-stimulated phosphoprotein (VASP) is a processive actin polymerase with roles in the control of cell shape and cell migration. Through interaction with the cytoskeletal adaptor protein Zyxin, VASP can localize to damaged stress fibers where it serves to repair and reinforce these structures. VASP localization is mediated by its N-terminal Ena/VASP homology (EVH1) domain, which binds to the (W/F)PxφP motif (most commonly occurring as FPPPP) found in cytoskeletal proteins such as vinculin, lamellipodin, and Zyxin. Sequentially close clusters of four or five of these motifs frequently occur, as in the proline rich region of Zyxin with four such motifs. This suggests that tetrameric VASP might bind very tightly to Zyxin through avidity, with all four EVH1 domains binding to a single Zyxin molecule. Here, quantitative nuclear magnetic resonance titration analysis reveals a dominant bivalent 1:1 (Zyxin:EVH1) interaction between the Zyxin proline rich region and the VASP EVH1 domain that utilizes the EVH1 canonical binding site and a novel secondary binding site on the opposite face of the EVH1 domain. We further show that binding to the secondary binding site is specifically inhibited by mutation of VASP EVH1 domain residue Y39 to E, which mimics Abl-induced phosphorylation of Y39. On the basis of these findings, we propose a model in which phosphorylation of Y39 acts as a stoichiometry switch that governs binding partner selection by the constitutive VASP tetramer. These results have broader implications for other multivalent VASP EVH1 domain binding partners and for furthering our understanding of the role of Y39 phosphorylation in regulating VASP localization and cellular function.

31P-dephased, 13C-detected REDOR for NMR crystallography at natural isotopic abundance.

Typically, the process of NMR-based structure determination relies on accurately measuring a large number of internuclear distances to serve as restraints for simulated annealing calculations. In solids, the rotational-echo double-resonance (REDOR) experiment is a widely used approach to determine heteronuclear dipolar couplings corresponding to distances usually in the range of 1.5-8Å. A challenge in the interpretation of REDOR data is the degeneracy of symmetric subunits in an oligomer or equivalent molecules in a crystal lattice, which produce REDOR trajectories that depend explicitly on two or more distances instead of one. This degeneracy cannot be overcome by either spin dilution (for molecules containing 31P, 19F and other highly abundant nuclei) or selective pulses (in the case where there is chemical shift degeneracy). For small, crystalline molecules, such as phosphoserine, we demonstrate that as many as five inter-molecular distances must be considered to model 31P-dephased REDOR data accurately. We report excellent agreement between simulation and experiment once lattice couplings, 31P chemical shift anisotropy, and radio-frequency field inhomogeneity are all taken into account. We also discuss the systematic inaccuracies that may result from approximations that consider only the initial slope of the REDOR trajectory and/or that utilize a two- or three-spin system. Furthermore, we demonstrate the applicability of 31P-dephased REDOR for validation or refinement of candidate crystal structures and show that this approach is especially informative for NMR crystallography of 31P-containing molecules.

Neighboring phosphoSer-Pro motifs in the undefined domain of IRAK1 impart bivalent advantage for Pin1 binding.

The peptidyl prolyl isomerase Pin1 has two domains that are considered to be its binding (WW) and catalytic (PPIase) domains, both of which interact with phosphorylated Ser/Thr-Pro motifs. This shared specificity might influence substrate selection, as many known Pin1 substrates have multiple sequentially close phosphoSer/Thr-Pro motifs, including the protein interleukin-1 receptor-associated kinase-1 (IRAK1). The IRAK1 undefined domain (UD) contains two sets of such neighboring motifs (Ser131/Ser144 and Ser163/Ser173), suggesting possible bivalent interactions with Pin1. Using a series of NMR titrations with 15N-labeled full-length Pin1 (Pin1-FL), PPIase, or WW domain and phosphopeptides representing the Ser131/Ser144 and Ser163/Ser173 regions of IRAK1-UD, bivalent interactions were investigated. Binding studies using singly phosphorylated peptides showed that individual motifs displayed weak affinities (> 100 µm) for Pin1-FL and each isolated domain. Analysis of dually phosphorylated peptides binding to Pin1-FL showed that inclusion of bivalent states was necessary to fit the data. The resulting complex model and fitted parameters were applied to predict the impact of bivalent states at low micromolar concentrations, demonstrating significant affinity enhancement for both dually phosphorylated peptides (3.5 and 24 µm for peptides based on the Ser131/Ser144 and Ser163/Ser173 regions, respectively). The complementary technique biolayer interferometry confirmed the predicted affinity enhancement for a representative set of singly and dually phosphorylated Ser131/Ser144 peptides at low micromolar concentrations, validating model predictions. These studies provide novel insights regarding the complexity of interactions between Pin1 and activated IRAK1, and more broadly suggest that phosphorylation of neighboring Ser/Thr-Pro motifs in proteins might provide competitive advantage at cellular concentrations for engaging with Pin1.

Isomerase-catalyzed binding of interleukin-1 receptor-associated kinase 1 to the EVH1 domain of vasodilator-stimulated phosphoprotein.

Interleukin-1 receptor-associated kinase 1 (IRAK1) is a crucial signaling kinase in the immune system, involved in Toll-like receptor signaling. Vasodilator-stimulated phosphoprotein (VASP) is a central player in cell migration that regulates actin polymerization and connects signaling events to cytoskeletal remodeling. A VASP­IRAK1 interaction is thought to be important in controlling macrophage migration in response to protein kinase C-ε activation. We show that the monomeric VASP EVH1 domain directly binds to the 168WPPPP172 motif in the IRAK1 undefined domain (IRAK1-UD) with moderate affinity (KDApp = 203 ± 3 µM). We further show that this motif adopts distinct cis and trans isomers for the Trp168­Pro169 peptide bond with nearly equal populations, and that binding to the VASP EVH1 domain is specific for the trans isomer, coupling binding to isomerization. Nuclear magnetic resonance line shape analysis and tryptophan fluorescence experiments reveal the complete kinetics and thermodynamics of the binding reaction, showing diffusion-limited binding to the trans isomer followed by slow, isomerization-dependent binding. We further demonstrate that the peptidyl-prolyl isomerase cyclophilin A (CypA) catalyzes isomerization of the Trp168­Pro169 peptide bond and accelerates binding of the IRAK1-UD to the VASP EVH1 domain. We propose that binding of IRAK1 to tetrameric VASP is regulated by avidity through the assembly of IRAK1 onto receptor-anchored signaling complexes and that an isomerase such as CypA may modulate IRAK1 signaling in vivo. These studies demonstrate a direct interaction between IRAK1 and VASP and suggest a potential mechanism for how this interaction might be regulated by both assembly of IRAK1 onto an activated signaling complex and PPIase enzymes.

Complete thermodynamic and kinetic characterization of the isomer-specific interaction between Pin1-WW domain and the amyloid precursor protein cytoplasmic tail phosphorylated at Thr668.

Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 µM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis-trans molecular switches and isomer-specific interactions involved in various biological processes.

Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis.

The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, [Formula: see text] and apparent Michaelis constants, [Formula: see text]. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific (13)C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide (13)C-(1)H constant time HSQC spectra to determine [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.

CRAC motif peptide of the HIV-1 gp41 protein thins SOPC membranes and interacts with cholesterol.

This study uses low-angle (LAXS) and wide-angle (WAXS) X-ray synchrotron scattering, volume measurements and thin layer chromatography to determine the structure and interactions of SOPC, SOPC/cholesterol mixtures, SOPC/peptide and SOPC/cholesterol/peptide mixtures. N-acetyl-LWYIK-amide (LWYIK) represents the naturally-occurring CRAC motif segment in the pretransmembrane region of the gp41 protein of HIV-1, and N-acetyl-IWYIK-amide (IWYIK), an unnatural isomer, is used as a control. Both peptides thin the SOPC bilayer by approximately 3 A, and cause the area/unit cell (peptide+SOPC) to increase by approximately 9 A2 from the area/lipid of SOPC at 30 degrees C (67.0+/-0.9 A2). Model fitting suggests that LWYIK's average position is slightly closer to the bilayer center than IWYIK's, and both peptides are just inside of the phosphate headgroup. Both peptides increase the wide-angle spacing d of SOPC without cholesterol, whereas with 50% cholesterol LWYIK increases d but IWYIK decreases d. TLC shows that LWYIK is more hydrophobic than IWYIK; this difference persists in peptide/SOPC 1:9 mole ratio mixtures. Both peptides counteract the chain ordering effect of cholesterol to roughly the same degree, and both decrease KC, the bending modulus, thus increasing the SOPC membrane fluidity. Both peptides nucleate crystals of cholesterol, but the LWYIK-induced crystals are weaker and dissolve more easily.

Partial molecular volumes of lipids and cholesterol.

Volumetric measurements are reported for fully hydrated lipid/cholesterol bilayer mixtures using the neutral flotation method. Apparent specific volume data were obtained with the lipids DOPC, POPC and DMPC at T=30 degrees Celsius, DPPC at 50 degrees Celsius, and brain sphingomyelin (BSM) at 45 and 24 degrees Celsius for mole fractions of cholesterol x from 0 to 0.5. Unlike previous cholesterol mixture studies, we converted our raw data to partial molecular volume V(L) of the lipid and V(C) of the cholesterol. The partial molecular volumes were constant for POPC and DOPC as x was varied, but had sharp breaks for the other lipids at values of x(C) near 0.25+/-0.05. Results for xx(C) the values of the partial molecular volumes of cholesterol clustered near 630+/-10A(3) in all the lipids when measured for T>T(M); we suggest that this is the most appropriate measure of the bare volume of cholesterol in lipid bilayers.