Reversed approach to S-farnesylation and S-palmitoylation: application to an efficient synthesis of the C-terminus of lipidated human N-ras hexapeptide

[reaction: see text] A general reversed approach is described to synthesize S-palmitoylated and S-farnesylated peptides via S(N)2 displacement of bromide by reaction of a thiol group containing lipid as nucleophile with bromoalanine-containing peptides as electrophile. By employing this approach, lipidated peptides, including characteristic partial structures of human Ras peptides, were synthesized in good yields. This method gives access to farnesylated, palmitoylated, and doubly lipidated peptides.     

Asymmetric ABC-triblock copolymer membranes induce a directed insertion of membrane proteins

Asymmetric molecules and materials provide an important basis for the organization and function of biological systems. It is well known that, for example, the inner and outer leaflets of biological membranes are strictly asymmetric with respect to lipid composition and distribution. This plays a crucial role for many membrane-related processes like carrier-mediated transport or insertion and orientation of integral membrane proteins. Most artificial membrane systems are, however, symmetric with respect to their midplane and membrane proteins are incorporated with random orientation. Here we describe a new approach to induce a directed insertion of membrane proteins into asymmetric membranes formed by amphiphilic ABC triblock copolymers with two chemically different water-soluble blocks A and C. In a comparative study we have reconstituted His-tag labeled Aquaporin 0 in lipid, ABA block copolymer, and ABC block copolymer vesicles. Immunolabeling, colorimetric, and fluorescence studies clearly show that a preferential orientation of the protein is only observed in the asymmetric ABC triblock copolymer membranes.     

Synthesis of lipidated eNOS peptides by combining enzymatic, noble metal- and acid-mediated protecting group techniques with solid phase peptide synthesis and fragment condensation in solution

Lipid-modified proteins play decisive roles in important biological processes such as signal transduction, organization of the cytoskeleton, and vesicular transport. Lipidated peptides embodying the characteristic partial structures of their parent lipidated proteins and semisynthetic proteins synthesized from such peptides are valuable tools for the study of these biological phenomena. We have developed an efficient synthesis strategy that allows for the synthesis of long multiply lipidated peptides embodying various side chain functional groups. The strategy was successfully applied in the synthesis of the N-terminal undetrigintapeptide of endothelial NO-synthase and related lipopeptides. Key elements of the synthesis strategy are the combined use of the enzyme-labile para-phenylacetoxybenzyloxycarbonyl (PhAcOZ) urethane as N-terminal blocking group, the Pd0-sensitive allyl ester as C-terminal protecting function and acid-labile side chain protecting groups for solution-phase synthesis of labile S-palmitoylated building blocks under the mildest conditions with solid-phase techniques and solution-phase fragment condensations. The successful synthesis of the triply lipidated 29-mer eNOS peptide convincingly demonstrates the full capacity of the protecting group methods.     

Oriented reconstitution of a membrane protein in a giant unilamellar vesicle: experimental verification with the potassium channel KcsA

We report a method for the successful reconstitution of the KcsA potassium channel with either an outside-out or inside-out orientation in giant unilamellar vesicles, using the droplet-transfer technique. The procedure is rather simple. First, we prepared water-in-oil droplets lined with a lipid monolayer. When solubilized KcsA was encapsulated in the droplet, it accumulated at monolayers of phosphatidylglycerol (PG) and phosphoethanolamine (PE) but not at a monolayer of phosphatidylcholine (PC). The droplet was then transferred through an oil/water interface having a preformed monolayer. The interface monolayer covered the droplet so as to generate a bilayer vesicle. By creating chemically different lipid monolayers at the droplet and oil/water interface, we obtained vesicles with asymmetric lipid compositions in the outer and inner leaflets. KcsA was spontaneously inserted into vesicles from the inside or outside, and this was accelerated in vesicles that contained PE or PG. Integrated insertion into the vesicle membrane and the KcsA orientation were examined by functional assay, exploiting the pH sensitivity of the opening of the KcsA when the pH-sensitive cytoplasmic domain (CPD) faces toward acidic media. KcsA loaded from the inside of the PG-containing vesicles becomes permeable only when the intravesicular pH is acidic, and the KcsA loaded from the outside becomes permeable when the extravesicular pH is acidic. Therefore, the internal or external insertion of KcsA leads to an outside-out or inside-out configuration so as to retain its hydrophilic CPD in the added aqueous side. The CPD-truncated KcsA exhibited a random orientation, supporting the idea that the CPD determines the orientation. Further application of the droplet-transfer method is promising for the reconstitution of other types of membrane proteins with a desired orientation into cell-sized vesicles with a targeted lipid composition of the outer and inner leaflets.     

Visualizing association of N-ras in lipid microdomains: influence of domain structure and interfacial adsorption

In this study, two-photon fluorescence microscopy on giant unilamellar vesicles and tapping-mode atomic force microscopy (AFM) are applied to follow the insertion of a fluorescently (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY) labeled and completely lipidated (hexadecylated and farnesylated) N-Ras protein into heterogeneous lipid bilayer systems. The bilayers consist of the canonical raft mixture 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin, and cholesterol, which-depending on the concentration of the constituents-separates into liquid-disordered (l(d)), liquid-ordered (l(o)), and solid-ordered (s(o)) phases. The results provide direct evidence that partitioning of N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation into the fluid lipid bilayers. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is l(d) > l(o) > s(o). Intriguingly, we detect, using the better spatial resolution of AFM, also a large proportion of the lipidated protein located at the l(d)/l(o) phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of the demixed phases. Such an interfacial adsorption effect may serve as an alternative vehicle for association processes of signaling proteins in membranes.     

The synthesis of acid- and base-labile lipopeptides on solid support

Lipidated peptides, including characteristic partial structures of human Ras proteins, were synthesized by means of a new solid-phase technique in 22-68 % yield. This technique gives access to farnesylated, palmitoylated, and doubly lipidated peptides as methyl esters or carboxylic acids carrying a fluorescent tag or a maleimide moiety for coupling to proteins. The peptide backbones were built up on the resin by using 9-fluorenylmethoxycarbonyl chemistry together with the oxidatively cleavable hydrazide linker. As a key step, the acid-labile farnesyl and basic-labile palmitoyl lipid groups were introduced onto the resin after the cleavage of appropriate acid- or reduction-sensitive protecting groups from the cysteine residues. Optional introduction of different fluorescent tags or a maleimide group into the peptide was followed by release of the resin-bound target peptide as the methyl ester or carboxylic acid by very mild copper(II)-mediated oxidation in slightly acidic or basic media. This new methodology should substantially facilitate the access to lipidated peptides for the study of important biological phenomena like biological signal transduction, localization, and vesicular transport.     

Whole-body rocking motion of a fusion peptide in lipid bilayers from size-dispersed 15N NMR relaxation

Biological membranes present a highly fluid environment, and integration of proteins within such membranes is itself highly dynamic: proteins diffuse laterally within the plane of the membrane and rotationally about the normal vector of this plane. We demonstrate that whole-body motions of proteins within a lipid bilayer can be determined from NMR (15)N relaxation rates collected for different-sized bicelles. The importance of membrane integration and interaction is particularly acute for proteins and peptides that function on the membrane itself, as is the case for pore-forming and fusion-inducing proteins. For the influenza hemagglutinin fusion peptide, which lies on the surface of membranes and catalyzes the fusion of membranes and vesicles, we found large-amplitude, rigid-body wobbling motions on the nanosecond time scale relative to the lipid bilayer. This behavior complements prior analyses where data were commonly interpreted in terms of a static oblique angle of insertion for the fusion peptide with respect to the membrane. Quantitative disentanglement of the relative motions of two interacting objects by systematic variation of the size of one is applicable to a wide range of systems beyond protein-membrane interactions.     

Fusogenic tilted peptides induce nanoscale holes in supported phosphatidylcholine bilayers

Tilted peptides are known to insert in lipid bilayers with an oblique orientation, thereby destabilizing membranes and facilitating membrane fusion processes. Here, we report the first direct visualization of the interaction of tilted peptides with lipid membranes using in situ atomic force microscopy (AFM) imaging. Phase-separated supported dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers were prepared by fusion of small unilamellar vesicles and imaged in buffer solution, in the absence and in the presence of the simian immunodeficiency virus (SIV) peptide. The SIV peptide was shown to induce the rapid appearance of nanometer scale bilayer holes within the DPPC gel domains, while keeping the domain shape unaltered. We attribute this behavior to a local weakening and destabilization of the DPPC domains due to the oblique insertion of the peptide molecules. These results were directly correlated with the fusogenic activity of the peptide as determined using fluorescently labeled DOPC/DPPC liposomes. By contrast, the nontilted ApoE peptide did not promote liposome fusion and did not induce bilayer holes but caused slight erosion of the DPPC domains. In conclusion, this work provides the first direct evidence for the production of stable, well-defined nanoholes in lipid bilayer domains by the SIV peptide, a behavior that we have shown to be specifically related to the tilted character of the peptide. A molecular mechanism underlying spontaneous insertion of the SIV peptide within lipid bilayers and the subsequent removal of bilayer patches is proposed, and its relevance to membrane fusion processes is discussed.     

Membrane localization and flexibility of a lipidated ras peptide studied by molecular dynamics simulations

Lipid-modified membrane-binding proteins are essential in signal transduction events of the cell, a typical example being the GTPase ras. Recently, membrane binding of a doubly lipid-modified heptapeptide from the C-terminus of the human N-ras protein was studied by spectroscopic techniques. It was found that membrane binding is mainly due to lipid chain insertion, but it is also favored by interactions between apolar side chains and the hydrophobic region of the membrane. Here, 10 explicit solvent molecular dynamics simulations for a total time of about 150 ns are used to investigate the atomic details of the peptide-membrane association. The 16:0 peptide lipid chains are more mobile than the 14:0 phospholipid chains, which is in agreement with (2)H NMR experiments. Peptide-lipid and peptide-solvent interactions, backbone and side-chain distributions, as well as the effects of lipidated peptide insertion onto the structure, and dynamics of a 1,2-dimyristoylglycero-3-phosphocholine bilayer are described. The simulation results validate the structural model proposed by the analysis of spectroscopic data and highlight the main aspects of the insertion mechanism. The peptide in the membrane is rather rigid over the simulation time scale of about 10 ns, but different partially extended conformations devoid of backbone hydrogen bonds are observed in different trajectories.     

Enzymatic Cell-Surface Decoration with Proteins using Amphiphilic Lipid-Fused Peptide Substrates

Lipid modification of proteins plays a significant role in the activation of cellular signals such as proliferation. Thus, the demand for lipidated proteins is rising. However, getting a high yield and purity of lipidated proteins has been challenging. We developed a strategy for modifying proteins with a wide variety of synthetic lipids using microbial transglutaminase (MTG), which catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and lysine (K) in the lipid-fused peptide. The synthesized lipid-G₃S-MRHKGS lipid (lipid: fatty acids, tocopherol, lithocholic acid, cholesterol) was successfully conjugated to a protein fused with LLQG (Q-tagged protein) by an MTG reaction, yielding >90 % conversion of the Q-tagged protein in a lipidated form. The purified lipid–protein conjugates were used for labeling the cell membrane in vitro, resulting in best-anchoring ability of cholesterol modification. Furthermore, in situ cell-surface decoration with the protein was established in a simple manner: subjection of cells to a mixture of cholesterol-fused peptides, Q-tagged proteins and MTG.     

Asymmetric droplet interface bilayers

In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. We and others recently developed the droplet interface bilayer (DIB), which is formed by connecting lipid monolayer-encased aqueous droplets submerged in an oil-lipid mixture. Here, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both alpha-helical and beta-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores.     

Binding of a truncated form of lecithin:retinol acyltransferase and its N- and C-terminal peptides to lipid monolayers

Lecithin:retinol acyltransferase (LRAT) is a 230 amino acid membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. A truncated form of LRAT (tLRAT), which contains the residues required for catalysis but which is lacking the N- and C-terminal hydrophobic segments, was produced to study its membrane binding properties. Measurements of the maximum insertion pressure of tLRAT, which is higher than the estimated lateral pressure of membranes, and the positive synergy factor a argue in favor of a strong binding of tLRAT to phospholipid monolayers. Moreover, the binding, secondary structure and orientation of the peptides corresponding to its N- and C-terminal hydrophobic segments of LRAT have been studied by circular dichroism and polarization-modulation infrared reflection absorption spectroscopy in monolayers. The results show that these peptides spontaneously bind to lipid monolayers and adopt an α-helical secondary structure. On the basis of these data, a new membrane topology model of LRAT is proposed where its N- and C-terminal segments allow to anchor this protein to the lipid bilayer.     

Invited Review Chemoenzymatic Synthesis of Lipidated Peptides

Summary.  This review highlights the use of enzymatic protecting group techniques in the synthesis of lipidated peptides. Lipidated proteins play key roles in signal transduction processes. Moreover, structurally well-defined peptides containing the characteristic linkage region of the peptide backbone with the lipid can provide valuable tools for the study of biological phenomena associated with these protein conjugates. The multifunctionality and pronounced lability towards acids and bases of such compounds render their synthesis a formidable challenge. However, the recent development of enzymatic protection groups provides an efficient access to these sensitive and biologically relevant peptide conjugates under particular mild conditions and with high selectivity. Received December 12, 1999. Accepted January 26, 2000     

Engineering bio-mimicking functional vesicles with multiple compartments for quantifying molecular transport

Controlled design of giant unilamellar vesicles under defined conditions has vast applications in the field of membrane and synthetic biology. Here, we bio-engineer bacterial-membrane mimicking models of controlled size under defined salt conditions over a range of pH. A complex bacterial lipid extract is used for construction of physiologically relevant Gram-negative membrane mimicking vesicles whereas a ternary mixture of charged lipids (DOPG, cardiolipin and lysyl-PG) is used for building Gram-positive bacterial-membrane vesicles. Furthermore, we construct stable multi-compartment biomimicking vesicles using the gel-assisted swelling method. Importantly, we validate the bio-application of the bacterial vesicle models by quantifying diffusion of chemically synthetic amphoteric antibiotics. The transport rate is pH-responsive and depends on the lipid composition, based on which a permeation model is proposed. The permeability properties of antimicrobial peptides reveal pH dependent pore-forming activity in the model vesicles. Finally, we demonstrate the functionality of the vesicles by quantifying the uptake of membrane-impermeable molecules facilitated by embedded pore-forming proteins. We suggest that the bacterial vesicle models developed here can be used to understand fundamental biological processes like the peptide assembly mechanism or bacterial cell division and will have a multitude of applications in the bottom-up assembly of a protocell.

Giant vesicle functional models mimicking a bacterial membrane under physiological conditions are constructed.     

Fusion of lipid vesicles with planar lipid bilayers induced by a combination of peptides

We studied the peptide-induced membrane fusion process between small unilamellar vesicles (SUVs) and supported planar bilayers (SPBs) with the aim of developing a method for incorporating membrane components into SPBs. As fusogenic peptides, two analogues of the N-terminal region of an influenza membrane fusion protein hemaggulutinin, anionic E5 and cationic K5, were synthesized, and the membrane fusion was investigated using SPB and SUVs composed of phosphatidylcholine from egg yolk (EggPC). We directly visualized the process of lipid transfer from SUVs to SPB by total internal reflection fluorescence (TIRF) microscopy. The transfer of fluorescent lipids was effectively induced only by the combination of two peptides. The TIRF microscopy observations of single SUV fusion events also revealed that lipid membranes from SUV could completely fuse into the SPB. However, the presence of single peptide (either E5 or K5) rather inhibited the lipid transfer, presumably due to the electrostatic repulsion between SUVs and SPB. The opposite effects induced by the peptides indicate the possibility for a designed application of two peptides as a means to control the membrane fusion spatially and temporally.     

Similar membrane affinity of mono- and Di-S-acylated ras membrane anchors: a new twist in the role of protein lipidation

The functionally required membrane attachment of Ras is achieved through an invariant isoprenylation of a C-terminal Cys, supplemented by further lipid modification of adjacent Cys residues by one (N-ras) or two (H-ras) palmitoyls. However, whether the triply lipidated membrane anchor of H-ras has a higher membrane affinity than its doubly lipidated counterpart, or whether the affinity contribution of the two palmitates and the farnesyl is additive, was not known. To address this issue, we carried out potential of mean force (PMF or free energy profile) calculations on a hexadecylated but nonpalmitoylated anchor (Cys186-HD), hexadecylated and monopalmitoylated anchors (Cys181-monopalmitate and Cys184-monopalmitate), and a nonlipid-modified anchor. We found that the overall insertion free energy follows the trend Cys181/Cys184-bipalmitate (wild type) approximately Cys181-monopalmitate > Cys184-monopalmitate > nonpalmitoylated anchor. Consistent with suggestions from recent cell biological experiments, the computed PMFs, coupled with structural analysis, demonstrate that membrane affinity of the Ras anchor depends on both the hydrophobicity of the palmitate and the prenyl groups and the spacing between them. The data further suggest that while Cys181-palmitate and Cys186-farnesyl together provide sufficient hydrophobic force for tight membrane binding, the palmitoyl at Cys184 is likely designed to serve another, presumably functional, role.     

Membrane interactions of host-defense peptides studied in model systems

Host-defense, antibiotic peptides are believed to generate their cytolytic effects by interacting with the membranes of bacterial cells. Direct analyses of peptide interactions with real cellular membranes are difficult, however, due to the high complexity of physiological membranes. This review summarizes experimental work aiming to understand peptide-membrane interactions and their relationships with the peptides' biological actions using specific model systems. Varied model assemblies have been constructed that generally aim to mimic the fundamental lipid bilayer organization of the membrane. The model systems we will describe include multilamellar and unilamellar vesicles, planar lipid bilayers, lipid monolayers and micelles, and colorimetric biomimetic membranes. The different artificial models have facilitated examination of specific biological or chemical parameters affecting peptide action, for example the effect of membrane lipid composition on peptide affinities and membrane penetration, the relationship between membrane fluidity and peptide interactions, the conformations of active peptides, and other factors. We evaluate the strengths and limitations of the various approaches, and point to future directions in the field.     

Exploiting Benzophenone Photoreactivity To Probe the Phospholipid Environment and Insertion Depth of the Cell-Penetrating Peptide Penetratin in Model Membranes

Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocation, thus implying that the peptide interacts with the lipid bilayer. Penetratin also crosses the membrane of artificial vesicles, depending on their phospholipid content. To evaluate the phospholipid preference of penetratin, as the first step of translocation, we exploited the benzophenone triplet kinetics of hydrogen abstraction, which is slower for secondary than for allylic hydrogen atoms. By using multilamellar vesicles of varying phospholipid content, we identified and characterized the cross-linked products by MALDI-TOF mass spectrometry. Penetratin showed a preference for negatively charged (vs. zwitterionic) polar heads, and for unsaturated (vs. saturated) and short (vs. long) saturated phospholipids. Our study highlights the potential of using benzophenone to probe the environment and insertion depth of membranotropic peptides in membranes.     

Exploiting Benzophenone Photoreactivity To Probe the Phospholipid Environment and Insertion Depth of the Cell‐Penetrating Peptide Penetratin in Model Membranes

Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocation, thus implying that the peptide interacts with the lipid bilayer. Penetratin also crosses the membrane of artificial vesicles, depending on their phospholipid content. To evaluate the phospholipid preference of penetratin, as the first step of translocation, we exploited the benzophenone triplet kinetics of hydrogen abstraction, which is slower for secondary than for allylic hydrogen atoms. By using multilamellar vesicles of varying phospholipid content, we identified and characterized the cross‐linked products by MALDI‐TOF mass spectrometry. Penetratin showed a preference for negatively charged (vs. zwitterionic) polar heads, and for unsaturated (vs. saturated) and short (vs. long) saturated phospholipids. Our study highlights the potential of using benzophenone to probe the environment and insertion depth of membranotropic peptides in membranes.