Model Membrane Vesicles Research Articles

The cation channel mucolipin-1 is a bifunctional protein that facilitates membrane remodeling via its serine lipase domain

Phospholipase modulators have been shown to affect the topology of lipid bilayers and the formation of tubulo-vesicular structures, but the specific endogenous phospholipases involved have yet to be identified. Here we show that TRPML1 (MLN1), a Ca(2+)-permeable channel, contributes to membrane remodeling through a serine lipase consensus domain, and thus represents a novel type of bifunctional protein. Remarkably, this serine lipase active site determines the ability of MLN1 to generate tubulo-vesicular extensions in mucolipin-1-expressing oocytes, human fibroblasts and model membrane vesicles. Our demonstration that MLN1 is involved in membrane remodeling and the formation of extensions suggests that it may play a role in the formation of cellular processes linked to the late endosome/lysosome (LE/L) pathway. MLN1 is absent or mutated in patients with mucolipidosis IV (MLIV), a lysosomal disorder with devastating neurological and other consequences. This study provides potential insight into the pathophysiology of MLIV.

Low pH-Induced Pore Formation by the T Domain of Botulinum Toxin Type A is Dependent upon NaCl Concentration

Botulinum neurotoxins (BoNTs) undergo low pH-triggered membrane insertion, resulting in the translocation of their light (catalytic) chains into the cytoplasm. The T (translocation) domain of the BoNT heavy chain is believed to carry out translocation. Here, the behavior of isolated T domain from BoNT type A has been characterized, both in solution and when associated with model membranes. When BoNT T domain prepared in the detergent dodecylmaltoside was diluted into aqueous solution, it exhibited a low pH-dependent conformational change below pH 6. At low pH the T domain associated with, and formed pores within, model membrane vesicles composed of 30 mol% dioleoylphosphatidylglycerol/70 mol% dioleoylphosphatidylcholine. Although T domain interacted with vesicles at low (50 mM) and high (400 mM) NaCl concentrations, the interaction required much less lipid at low salt. However, even at high lipid concentrations pore formation was much more pronounced at low NaCl concentrations than at high NaCl concentration. Increasing salt concentration after insertion in the presence of 50 mM NaCl did not decrease pore formation. A similar effect of NaCl concentration upon pore formation was observed in vesicles composed solely of dioleoylphosphatidylcholine, showing that the effect of NaCl did not solely involve modulation of electrostatic interactions between protein and anionic lipids. These results indicate that some feature of membrane-bound T domain tertiary structure critical for pore formation is highly dependent upon salt concentration.

Interaction of an Endosomolytic Polyamidoamine ISA23 with Vesicles Mimicking Intracellular Membranes: A SANS/EPR Study

The mechanism of ISA23 · HCl interaction with model membrane vesicles (80-100 nm in diameter) was investigated using EPR in conjunction with SANS. For EPR, 16-DSE was dissolved in the vesicle membrane to measure its dynamics and polarity, whereas a spin-labeled (Tempo)-ISA 23 analogue was used to give a measure of the polymer flexibility. When ISA23 was added to the external vesicle surface, no interaction was found. This observation conflicts with the reported ability to lyse RBC, but is in agreement with recent studies that showed no effect on membrane permeability when a PAA was added to an incubation medium containing isolated lysosomal vesicles. The vesicle-mimetic models used here provide a new and useful tool for studying endosomolytic polymer/membrane interactions.

The Use of Time-Resolved Fluorescence Anisotropy to Reveal Domain Structures in Model Membrane Vesicles: Prospects for Applications to Cell Membranes

Cell membranes are thought to consist of structurally distinct regions within which reside functionally-interacting cell membrane components. Although much research has been directed toward the detection of these regions, variously known as domains or rafts, they still remain elusive entities. A number of techniques have used probes that tend to reside in these regions and using this approach structures have been identified using florescence spectroscopic approaches. Here we have investigated the motion of the probes rather that their presence to identify the presence of domain regions using giant unilamellar vesicles (GUV) to model cell membranes. Using the electroformation method, GUV were made using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin and cholesterol in various proportions. The fluorescent probes, diphenyhexatriene (DPH), 1-acyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (C6-NBD-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) were pre-incorporated in the GUV. The time-resolved anisotropy of the probes was determined using 2-photon excitation from a Ti-sapphire laser (180 fs pulses at 75 MHz and 750 nm) coupled to a microscope. Excitation was performed with an incident vertically polarized light and a Y-output acquisition, generating simultaneous vertical and horizontal detection, using a multi-channel photon multiplier tube. The output signal was fed through a Becker and Hickl SPC-830 module and anisotropy analysis software. The set up allows for picosecond time resolution as well as diffraction limited image resolution needed for the domain identification. It was found that each probe had two distinct motions corresponding to different orientations and locations in single-component vesicles, as known for DPH, but not previously observed for the NBD probes. Using polarization direction selectivity these two locations could be separately examined using the current development. The data shows that NBD selectively locates into domain regions in three-component vesicle systems.

Solid-State NMR Structural Studies of Alzheimer's Disease Aβ(1-42) And Aβ(1-40) In Phospholipid Bilayers

Amyloid-β peptides, Aβ(1-42) and Aβ(1-40) are believed to cause loss of nerve cell function in individuals suffering from Alzheimer's disease, where evidence suggests that interaction with the cell membrane correlates strongly with cytotoxicity. Previous studies report a range of different but plausible structures, which depend on the molecular environment of the peptide. This sensitivity to sample preparation suggests that the structure relevant to disease is found in lipid bilayer membranes. While model membrane vesicles are too large to be studied by conventional solution NMR, solid-state NMR is one of the few technologies available to study such systems. We present recent magic angle spinning NMR measurements which suggest a novel peptide structure in a lipid bilayer environment: (i) rotational-echo double-resonance (REDOR) distance measurements between selectively enriched 13C-carbonyl and 15N-amide positions constrain dihedral angles of intervening residues and suggest that at least part of the Aβ(1-42) peptide folds into a β-sheet like conformation, in contrast to the helical and coiled structures in previous reports; and (ii) double quantum filtered DRAWS measurements indicate that the extended strand does not assemble into an in-register parallel sheet as reported for amyloid fibrils. Additional static NMR experiments (sensitivity enhance PISEMA) of 15N labelled Aβ(1-40) in mechanically aligned phospholipid bilayers and magnetically oriented bicelles indicate that the peptide has multiple conformations in model membranes.

An efficient synthesis of cholesterol formate

Cholesterol formate is an important compound in the chemistry of steroids. It is used in biochemical investiga tions of model membrane vesicles.1 Recently, cholesterol formate has been isolated from the red alga Grateloupia turuturu Yamada and this may be of chemotaxonomic sig nificance for this organism.2 This compound is employed as a component in liquid crystal optical filters.3 Formyl protection is widely used in cholesterol chemistry because of its selective removal under mild conditions (K2CO3, methanol, 20 °C) in the presence of other ester protecting groups.4 That is why a search for new convenient routes to cholesterol formate is of relentless interest. Common reagents employed for O formylation of cho lesterol include DMF—benzoyl chloride,5 DMF—tri arylphosphine—halogen,6 EtOCHO—Cu(NO3)2•3H2O, 7 MeOCHO—Ph3P—CBr4, 8 CCl3CHO—K2CO3, 9 EtOCHO—PCl3—SiO2), 10 and DMF—POCl3. 11 Usual ly, they are used in large molar excesses with respect to cholesterol. For instance, formylation of cholesterol with DMF—POCl3 (the Vilsmeier complex), which is poorly reproducible and inefficient for alcohols,5 is carried out in the presence of its four to fivefold molar excess (~20 °C, 3 h, 64% yield).11 In our recent synthesis of 1 vinylpyrrole 2 carbalde hydes,12 we found that the complex DMF—oxalyl chlo ride is a milder and more efficient formylating reagent than the classic Vilsmeier system. Our investigations in the borderland between the chem istry of pyrroles and steroids,13 including novel approach es to their vinylation14 and formylation,12,15 led us to a con venient and efficient route to cholesterol formate. At a molar ratio cholesterol : DMF—oxalyl chloride of 1 : 1.5, the re action in CH2Cl2 at ~20 °C is completed in 40 min to give the target product in 97% yield (Scheme 1).

The Effect of Hydrophilic Substitutions and Anionic Lipids upon the Transverse Positioning of the Transmembrane Helix of the ErbB2 (neu) Protein Incorporated into Model Membrane Vesicles

The sequence of the transmembrane (TM) helix of ErbB2, a member of the epidermal growth factor receptor (ErbB) family, can influence its activity. In this report, the sequence and lipid dependence of the transverse position of a model-membrane-inserted peptides containing the ErbB2 TM helix and some of the juxtamembrane (JM) residues were studied. For the ErbB2 TM helix inserted into phosphatidylcholine vesicles, the activating V664E mutation was found to induce a transverse shift involving the movement of the E residue toward the membrane surface. This shortened the effective length of the TM-spanning portion of the sequence. The transverse shift was observed with the E664 residue in both the uncharged and charged states, but the extent of the shift was larger when the E residue was charged. When a series of hydrophilic residues was substituted for V664, the resulting transverse shifts at pH 7.0 decreased in the order D,H>E>Q>K>G>V. Except for His, this order is strongly correlated to that reported for the degree to which these substitutions induce cellular transformation when introduced into full-length ErbB2. To examine the effect of lipid on transverse shift, we studied the uncharged V664Q mutation. The presence of 20% of the anionic lipid DOPS (dioleoylphosphatidylserine) in the model membrane vesicles, which introduces a physiologically relevant level of anionic lipid, did not affect the degree of transverse shift. However, in the case of a peptide containing a V674Q substitution, in which the Q is closer to the C-terminus of the ErbB2 TM helix than the N-terminus, transverse shift was suppressed in vesicles containing 20% DOPS. This suggests that the interaction of the cationic JM residues flanking the C-terminus of the ErbB2 TM helix interact with anionic lipids to anchor the C-terminal end of the TM helix. This anchoring site may act as a pivot that amplifies transverse movements of the ErbB2 TM segment to induce a large swinging-type motion in the extracellular domain of the protein, affecting ErbB2 activity. Interactions interrupting C-terminal JM residue association with anionic lipid might partly impact ErbB2 activity by disrupting this pivoting.

Effect of Lipid Composition on the Topography of Membrane-Associated Hydrophobic Helices: Stabilization of Transmembrane Topography by Anionic Lipids

To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition on the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine ∼ 6:4 POPC:cholesterol < POPC ∼ dioleoylphosphatidylcholine (DOPC) < 1,2-dioleoyl- sn-glycero-3-[phospho- rac-(1-glycerol)] sodium salt (DOPG) ≤ 1,2-dioleoyl- sn-glycero-3-[phospho- l-serine] sodium salt (DOPS), indicating that the anionic lipids DOPG and DOPS most strongly stabilized the TM configuration. TM stabilization was near maximal at 20–30 mol% anionic lipid, which are physiologically relevant values. TM stabilization by anionic lipid was observed for hydrophobic sequences with a diverse set of sequences (including polyAla), diverse lengths (from 12 to 22 residues), and various cationic flanking residues (H, R, or K), but not when the flanking residues were uncharged. TM stabilization by anionic lipid was also dependent on the number of cationic residues flanking the hydrophobic sequence, but was still significant with only one cationic residue flanking each end of the peptide. These observations are consistent with TM-stabilizing effects being electrostatic in origin. However, Trp located more deeply in DOPS vesicles relative to DOPG vesicles, and peptides in DOPS vesicles showed increased helix formation relative to DOPG and all other lipid compositions. These observations fit a model in which DOPS anchors flanking residues near the membrane surface more strongly than does DOPG and/or increases the stability of the TM state to a greater degree than DOPG. We conclude that anionic lipids can have significant and headgroup structure-specific effects upon membrane protein topography.

The Control of Transmembrane Helix Transverse Position in Membranes by Hydrophilic Residues

The ability of hydrophilic residues to shift the transverse position of transmembrane (TM) helices within bilayers was studied in model membrane vesicles. Transverse shifts were detected by fluorescence measurements of the membrane depth of a Trp residue at the center of a hydrophobic sequence. They were also estimated from the effective length of the TM-spanning sequence, derived from the stability of the TM configuration under conditions of negative hydrophobic mismatch. Hydrophilic residues (at the fifth position in a 21-residue hydrophobic sequence composed of alternating Leu and Ala residues and flanked on both ends by two Lys) induced transverse shifts that moved the hydrophilic residue closer to the membrane surface. At pH 7, the dependence of the extent of shift upon the identity of the hydrophilic residue increased in the order: L < G∼Y∼T < R∼H < S < P < K < E∼Q < N < D. By varying pH, shifts with ionizable residues fully charged or uncharged were measured, and the extent of shift increased in the order: L < G∼Y∼H o∼T < E o∼R < S < P < K +< Q∼D o∼H + < N∼E – < D –. The dependence of transverse shifts upon hydrophilic residue identity was consistent with the hypothesis that shift magnitude is largely controlled by the combination of side chain hydrophilicity, ionization state, and ability to position polar groups near the bilayer surface (snorkeling). Additional experiments showed that shift was also modulated by the position of the hydrophilic residue in the sequence and the hydrophobicity of the sequence moved out of the bilayer core upon shifting. Combined, these studies show that the insertion boundaries of TM helices are very sensitive to sequence, and can be altered even by weakly hydrophilic residues. Thus, many TM helices may have the capacity to exist in more than one transverse position. Knowledge of the magnitudes of transverse shifts induced by different hydrophilic residues should be useful for design of mutagenesis studies measuring the effect of transverse TM helix position upon function.

Laurdan fluorescence spectroscopy in the thylakoid bilayer: The effect of violaxanthin to zeaxanthin conversion on the galactolipid dominated lipid environment

Laurdan (6-lauroyl-2-dimethylaminonaphthalene) fluorescence spectroscopy has been applied to probe the physical status of the thylakoid membrane upon conversion of violaxanthin to zeaxanthin. So far, only phospholipid-dominated membranes have been studied by this method and hereby we report the first use of laurdan in mono- and digalactosyldiacylglycerol-dominated membrane systems. The generalised polarisation (GP) of laurdan was used as a measure of the structural effect of xanthophyll cycle pigments in isolated spinach ( Spinacia oleracea) thylakoids and in model membrane vesicles composed of chloroplast galactolipids. Higher GP values indicate a membrane in a more ordered structure, whereas lower GP values point to a membrane in a less ordered fluid phase. The method was used to probe the effect of violaxanthin and zeaxanthin in thylakoid membranes at different temperatures. At 4, 25 and 37 °C the GP values for dark-adapted thylakoids in the violaxanthin-form were 0.55, 0.28 and 0.26. After conversion of violaxanthin to zeaxanthin, at the same temperatures, the GP values were 0.62, 0.36 and 0.34, respectively. GP values increased gradually upon conversion of violaxanthin to zeaxanthin. Similar results were obtained in the liposomal systems in the presence of these xanthophyll cycle pigments. We conclude from these results that the conversion of violaxanthin to zeaxanthin makes the thylakoid membrane more ordered.

Membrane Topography of the Hydrophobic Anchor Sequence of Poliovirus 3A and 3AB Proteins and the Functional Effect of 3A/3AB Membrane Association upon RNA Replication

Replication of poliovirus RNA takes place on the cytoplasmic surface of membranous vesicles that form after infection of the host cell. It is generally accepted that RNA polymerase 3D(pol) interacts with membranes in a complex with viral protein 3AB, which binds to membranes by means of a hydrophobic anchor sequence that is located near the C-terminus of the 3A domain. In this study, we used fluorescence and fluorescence quenching methods to define the topography of the anchor sequence in the context of 3A and 3AB proteins inserted in model membranes. Mutants with a single tryptophan near the center of the anchor sequence but lacking Trp elsewhere in 3A/3AB were constructed which, after the emergence of suppressor mutations, replicated well in HeLa cells. When a peptide containing the mutant anchor sequence was incorporated in model membrane vesicles, measurements of Trp depth within the lipid bilayer indicated formation of a transmembrane topography. However, rather than the 22-residue length predicted from hydrophobicity considerations, the transmembrane segment had an effective length of 16 residues, such that Gln64 likely formed the N-terminal boundary. Analogous experiments using full-length proteins bound to preformed model membrane vesicles showed that the anchor sequence formed a mixture of transmembrane and nontransmembrane topographies in the 3A protein but adopted only the nontransmembrane configuration in the context of 3AB protein. Studies of the function of 3A/3AB inserted into model membrane vesicles showed that membrane-bound 3AB is highly efficient in stimulating the activity of 3D(pol) in vitro while membrane-bound 3A totally lacks this activity. Moreover, in vitro uridylylation reactions showed that membrane-bound 3AB is not a substrate for 3D(pol), but free VPg released by cleavage of 3AB with proteinase 3CD(pro) could be uridylylated.

Topography of Diphtheria Toxin A Chain Inserted into Lipid Vesicles

The membrane-inserting T domain of diphtheria toxin aids the low-pH-triggered translocation of the catalytic A chain of the toxin across endosomal membranes. To evaluate the role of the isolated A chain in translocation, the topography of isolated A chain inserted into model membrane vesicles was investigated using a mixture either of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) or of dimyristoleoylphosphatidylcholine (DMoPC) and DOPG. The latter mixture was previously found to promote deep insertion of the T domain. A series of single Cys mutants along the A chain sequence were labeled with bimane or BODIPY groups. After A chain insertion into model membranes, the location of these groups within the lipid bilayer was determined via bimane fluorescence emission lambda(max), binding of externally added anti-BODIPY antibodies, and a novel technique involving the comparison of the quenching of bimane fluorescence by aqueous iodide and membrane-associated 10-doxylnonadecane. The results show that in both DOPC- and DMoPC-containing bilayers, membrane-inserted residues all along the A chain sequence occupy shallow locations that are relatively exposed to the external solution. There were only small differences between A chain topography in the two different types of lipid mixtures. However, the behavior of the A chain in the two different lipid mixtures was distinct in that it strongly oligomerized in DMoPC-containing vesicles as judged by Trp fluorescence. In addition, A chain selectively induced fusion of the DMoPC-containing vesicles, and this may aid oligomerization by increasing the A chain/vesicle ratio. Fusion may also explain why A chain also selectively induced leakage of the contents of DMoPC-containing vesicles. We propose that isolated A chain is unlikely to be inserted in a transmembrane orientation, and thus its interaction with the T domain is likely to be critical for properly orienting the A chain within the bilayer in a fashion that allows translocation.

Analyzing topography of membrane-inserted diphtheria toxin T domain using BODIPY-streptavidin: at low pH, helices 8 and 9 form a transmembrane hairpin but helices 5-7 form stable nonclassical inserted segments on the cis side of the bilayer.

Low pH-induced membrane insertion by diphtheria toxin T domain is crucial for A chain translocation into the cytoplasm. To define the membrane topography of the T domain, the exposure of biotinylated Cys residues to the cis and trans bilayer surfaces was examined using model membrane vesicles containing a deeply inserted T domain. To do this, the reactivity of biotin with external and vesicle-entrapped BODIPY-labeled streptavidin was measured. The T domain was found to insert with roughly 70-80% of the molecules in the physiologically relevant orientation. In this orientation, residue 349, located in the loop between hydrophobic helices 8 and 9, was exposed to the trans side of the bilayer, while other solution-exposed residues along the hydrophobic helices 5-9 region of the T domain located near the cis surface. A protocol developed to detect the movement of residues back and forth across the membranes demonstrated that T domain sequences did not rapidly equilibrate between the cis and the trans sides of the bilayer. Binding streptavidin to biotinylated residues prior to membrane insertion only inhibited T domain pore formation for residues in the loop between helices 8 and 9. Pore formation experiments used an approach avoiding interference from transient membrane defects/leakage that may occur upon the initial insertion of protein. Combined, these results indicate that at low pH hydrophobic helices 8 and 9 form a transmembrane hairpin, while hydrophobic helices 5-7 form a nonclassical deeply inserted nontransmembraneous state. We propose that this represents a novel pre-translocation state that is distinct from a previously defined post-translocation state.

The effect of interactions involving ionizable residues flanking membrane-inserted hydrophobic helices upon helix-helix interaction.

We examined the effect of ionizable residues at positions flanking the hydrophobic core of helix-forming polyLeu peptides upon helix-helix interactions within model membrane vesicles composed of dioleoylphosphatidylcholine. The peptides studied were flanked on both the N and C termini either by two Lys (K(2)-flanked peptide), one Lys plus one Asp (DK-flanked peptide), or one Lys plus three Asp (KD(3)-flanked peptide). The fluorescence of a Trp residue positioned at the center of the hydrophobic sequence was used to evaluate peptide behavior. As judged by the concentration dependence of the maximum wavelength of Trp emission, there was significant oligomerization of the KD(3)- and DK-flanked peptides, but not the K(2)-flanked peptide, at neutral pH. At neutral pH mixtures of K(2)- and KD(3)-flanked peptides associated with each other, but mixtures of the K(2)- and DK-flanked peptides did not. Oligomerization by the DK- and KD(3)-flanked peptides decreased under low pH conditions in which the Asp residues were protonated. Additional experiments showed that at neutral pH the KD(3)-flanked peptide showed an increased tendency to oligomerize when as little as 10-15 mol % of an anionic lipid, phosphatidylglycerol, was present. The behavior of the other peptides was not strongly influenced by phosphatidylglycerol. These results can largely be explained by modulation of helix-helix interactions via electrostatic interactions involving the helix-flanking ionizable residues. Such interactions may influence membrane protein folding. The self-association of anionic KD(3)-flanked peptides suggests that additional interactions involving charged residues also can modulate helix-helix association.

Cumulative effects of amino acid substitutions and hydrophobic mismatch upon the transmembrane stability and conformation of hydrophobic alpha-helices.

The effects of amino acid substitutions upon the behavior of poly(Leu)-rich alpha-helices inserted into model membrane vesicles were investigated. One or two consecutive Leu residues in the hydrophobic core of the helix were substituted with A, F, G, S, D, K, H, P, GG, SS, PG, PP, KK, or DD residues. A Trp placed at the center of the sequence allowed assessment of peptide behavior via fluorescence emission lambda(max) and dual quenching analysis of Trp depth [Caputo, G. A., and London, E. (2003) Biochemistry 42, 3265-3274]. In vesicles composed of dioleoylphosphatidylcholine (DOPC), all of the peptides with single substitutions adopted a transmembrane (TM) state. Experiments were also performed in thicker bilayers composed of dierucoylphosphatidylcholine (DEuPC). In DEuPC vesicles TM states were destabilized by mismatch between helix length and bilayer thickness. Nevertheless, in DEuPC vesicles TM states were still prevalent for peptides with single substitutions, although less so for peptides with P, K, H, or D substitutions. In contrast to single substitutions, certain consecutive double substitutions strongly interfered with formation of TM states. In both DOPC and DEuPC vesicles DD and KK substitutions abolished the normal TM state, but GG and SS substitutions had little effect. In even wider bilayers, a SS substitution reduced the formation of a TM state. A peptide with a PP substitution maintained the TM state in DOPC vesicles, but in DEuPC vesicles the level of formation of the TM state was significantly reduced. Upon disruption of normal TM insertion peptides moved close to the bilayer surface, with the exception of the KK-substituted peptide in DOPC vesicles, which formed a truncated TM segment. These studies begin to provide a detailed relationship between sequence and the stability of TM insertion and show that the influence of insertion-destabilizing residues upon hydrophobic helices can be strongly modulated by properties such as mismatch. For certain helix-forming hydrophobic sequences, sensitivity to lipid structure may be sufficient to induce large conformational changes in vivo.

Lipid II induces a transmembrane orientation of the pore-forming peptide lantibiotic nisin.

Nisin is an antimicrobial peptide produced by Lactococcus lactis and used as a food preservative in dairy products. The peptide kills Gram-positive bacteria via the permeabilization of the membrane, most probably via pore formation using the cell wall precursor Lipid II as its docking molecule. In this study, site-directed tryptophan spectroscopy was used to determine the topology of nisin in the Lipid II containing membrane, as a start to elucidate the mechanism of targeted pore formation. Three single tryptophan mutants were used, which are viable representatives of the wild-type peptide. The emission spectra of tryptophans located at the N-terminus, the center, and the C-terminus as well as quenching by acrylamide and spin-labeled lipids were investigated using model membrane vesicles composed of DOPC containing 1 mol % Lipid II. Nisin was shown to adopt an orientation where the most probable position of the N-terminus was found to be near the Lipid II headgroup at the bilayer surface, the position of the center of nisin was in the middle of the phospholipid bilayer, and the C-terminus was located near the interface between the headgroups and acyl chain region. These results were used to propose a model for the orientation of nisin in Lipid II containing membranes. Our findings demonstrated that Lipid II changes the overall orientation of nisin in membranes from parallel to perpendicular with respect to the membrane surface. The stable transmembrane orientation of nisin in the presence of Lipid II might allow us to determine the structure of the nisin-Lipid II pores in the lipid bilayer.

Influence of gp41 fusion peptide on the kinetics of poly(ethylene glycol)-mediated model membrane fusion.

The fusion peptide of the HIV fusion protein gp41 is required for viral fusion and entry into a host cell, but it is unclear whether this 23-residue peptide can fuse model membranes. We address this question for model membrane vesicles in the presence and absence of aggregating concentrations of poly(ethylene glycol) (PEG). PEG had no effect on the physical properties of peptide bound to membranes or free in solution. We tested for fusion of both highly curved and uncurved PC/PE/SM/CH (35:30:15:20 mol %) vesicles and highly curved PC/PE/CH (1:1:1) vesicles treated with peptide in the presence and absence of PEG. Fusion was never observed in the absence of PEG, although high peptide concentrations led to aggregation and rupture, especially in unstable PC/PE/CH (1:1:1) vesicles. When 5 wt % PEG was present to aggregate vesicles, peptide enhanced the rate of lipid mixing between curved PC/PE/SM/CH vesicles in proportion to the peptide concentration, with this effect leveling off at peptide/lipid (P/L) ratios approximately 1:200. Peptide produced an even larger effect on the rate of contents mixing but inhibited contents mixing at P/L ratios >1:200. No fusion enhancement was seen with uncurved vesicles. The rate of fusion was also enhanced by the presence of hexadecane, and peptide-induced rate enhancement was not observed in the presence of hexadecane. We conclude that gp41 fusion peptide does not induce vesicle fusion at subrupturing concentrations but can enhance fusion between highly curved vesicles induced to fuse by PEG. The different effects of peptide on the rates of lipid mixing and fusion pore formation suggest that, while gp41 fusion peptide does affect hemifusion, it mainly affects pore formation.

Influence of lipid composition on physical properties and peg-mediated fusion of curved and uncurved model membrane vesicles: "nature's own" fusogenic lipid bilayer.

Poly(ethylene glycol) (PEG)-mediated fusion of phosphatidylcholine model membranes has been shown to mimic the protein-mediated biomembrane process [Lee, J., and Lentz, B. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9274-9279]. Unlike the simple model membranes used in this earlier study, the lipid composition of fusogenic biomembranes is quite complex. The purpose of this paper was to examine PEG-mediated fusion of highly curved (SUV) and largely uncurved (LUV) membrane vesicles composed of different lipids in order to identify lipid compositions that produce highly fusogenic membranes. Starting with liposomes composed of five lipids with different physical properties, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), bovine brain sphingomyelin (SM), and cholesterol (CH), we systematically varied the composition and tested for the extent of PEG-mediated fusion after 5 min of treatment. We found that a vesicle system composed of four lipids, DOPC/DOPE/SM/CH, fused optimally at a 35/30/15/20 molar ratio. Each lipid seemed to play a part in optimizing the membrane for fusion. PE disrupted outer leaflet packing as demonstrated with TMA-DPH lifetime, C(6)-NBD-PC partitioning, and DPH anisotropy measurements, and thus significantly enhanced fusion and rupture, without significantly altering interbilayer approach (X-ray diffraction). An optimal ratio of PC/PE (35/30) produced a balance between fusion and rupture. CH and SM, when present at an optimal ratio of 3/4 in vesicles containing the optimal PC/PE ratio, reduced rupture without significantly reducing fusion. This optimal CH/SM ratio also enhanced outer leaflet packing, suggesting that fusion is dependent not only on outer leaflet packing but also on the properties of the inner leaflet. Addition of CH without SM enhanced rupture relative to fusion, while SM alone reduced both rupture and fusion. The optimal lipid composition is very close to the natural synaptic vesicle composition, suggesting that the synaptic vesicle composition is optimized with respect to fusogenicity.

Kinetic model of protein-mediated ligand transport: influence of soluble binding proteins on the intermembrane diffusion of a fluorescent fatty acid.

The mechanism (or mechanisms) whereby fatty acids and other amphipathic compounds are transported from the plasma membrane to intracellular sites of biotransformation remains poorly defined. In an attempt to better characterize the role of cytosolic binding proteins in this process, a kinetic model of intermembrane ligand transport was developed in which diffusional transfer of ligand between membrane and protein is assumed. The model was tested by utilizing stopped-flow techniques to monitor the transfer of the fluorescent fatty acid analogue, 12-anthroyloxy stearate (12-AS), between model membrane vesicles. Studies were conducted in the presence or absence of bovine serum albumin (BSA), liver fatty acid-binding protein (L-FABP), and intestinal fatty acid-binding protein (I-FABP) in order to determine the effect of soluble proteins on the rate of intermembrane ligand transfer. As predicted by the model, the initial velocity of 12-AS arrival at the acceptor membrane increases in an asymptotic manner with the acceptor concentration. Furthermore, probe transfer velocity was found to decline asymptotically with increasing concentrations of BSA or L-FABP, proteins that exhibit diffusional transfer kinetics. This observation was found to hold true independent of whether donor or acceptor vesicles were preequilibrated with the protein. In contrast, 12-AS transfer velocity exhibited a linear correlation with the concentration of I-FABP, a protein that is thought to transport fatty acids, at least in part, via a collisional mechanism. Taken together, these findings validate the derived kinetic model of protein-mediated ligand transport and further suggest that the mechanism of ligand-protein interaction is a key determinant of the effect of cytosolic proteins on intracellular ligand diffusion.

The effects of polar and/or ionizable residues in the core and flanking regions of hydrophobic helices on transmembrane conformation and oligomerization.

To explore the influence of amino acid composition on the behavior of membrane-inserted alpha-helices, we examined the behavior of Lys-flanked polyleucyl (pLeu) helices containing a single polar/ionizable residue within their hydrophobic core. To evaluate the location of the helices within the membrane by fluorescence, each contained a Trp residue at the center of the sequence. When incorporated into dioleoylphosphatidylcholine (DOPC) model membrane vesicles, pLeu helices with or without a single Ser, Asn, Lys, or Asp residue in the hydrophobic core maintained a transmembrane state (named the N state) at neutral and acidic pH. In this state, the central Trp exhibited highly blue-shifted fluorescence, and fluorescence quenching by nitroxide-labeled lipids showed it located at the bilayer center. A state in which Trp fluorescence red-shifted by several nanometers (named the B state) was observed above pH 10-11. B state formation appears to result from deprotonation of the flanking Lys residues. Despite the red shift in Trp emission, fluorescence quenching showed that in the B state the Trp at most is only slightly shallower than in the N state, suggesting the B state also is a transmembrane or near-transmembrane structure. The B state is characterized by increased helix oligomerization, as shown by the dependence of Trp lambda(max) on the concentration of the peptide within the bilayer at high pH. The pLeu peptide with a Asp residue in the core underwent a pH-dependent transition at a lower pH than the other peptides (pH 8-9). At high pH, it exhibited both a more highly red-shifted fluorescence and shallower Trp location than the other peptides. This state (named the S state) did not exhibit a concentration-dependent Trp lambda(max). We attribute S state behavior to the formation of a charged Asp residue at high pH, and a consequent movement of the Asp toward the membrane surface, resulting in the formation of a nontransmembrane state. We conclude that a polar or ionizable residue can readily be tolerated in a single transmembrane helix, but that the charges on ionizable residues in the core and regions flanking the helix significantly modulate the stability of transmembrane insertion and/or helix-helix association.