Membrane interaction of neuropeptide Y detected by EPR and NMR spectroscopy (original) (raw)
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The interaction of neuropeptide Y with negatively charged and zwitterionic phospholipid membranes
European Biophysics Journal, 2009
The interaction of the 36 amino acid neuropeptide Y (NPY) with liposomes was studied using the intrinsic tyrosine fluorescence of NPY and an NPY fragment comprising amino acids 18-36. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the experimentally measured binding curves, the standard Gibbs free energy for the peptide transfer from aqueous solution to the lipid membrane was calculated to be around -30 kJ/mol for membrane mixtures containing physiological amounts of acidic lipids at pH 5. The effective charge of the peptide depends on the pH of the buffer and is about half of its theoretical net charge. The results were confirmed using the fluorescence of the NPY analogue [Trp 32 ]-NPY. Further, the position of NPY's a-helix in the membrane was estimated from the intrinsic tyrosine fluorescence of NPY, from quenching experiments with spin-labelled phospholipids using [Trp 32 ]-NPY, and In memoriam Dr. Olaf Zschörnig (1958.
Biophysical Journal, 1996
We measured directly the binding of Lys3, Lys5, and Lys7 to vesicles containing acidic phospholipids. When the vesicles contain 33% acidic lipids and the aqueous solution contains 100 mM monovalent salt, the standard Gibbs free energy for the binding of these peptides is 3, 5, and 7 kcal/mol, respectively. The binding energies decrease as the mol% of acidic lipids in the membrane decreases and/or as the salt concentration increases. Several lines of evidence suggest that these hydrophilic peptides do not penetrate the polar headgroup region of the membrane and that the binding is mainly due to electrostatic interactions. To calculate the binding energies from classical electrostatics, we applied the nonlinear Poisson-Boltzmann equation to atomic models of the phospholipid bilayers and the basic peptides in aqueous solution. The electrostatic free energy of interaction, which arises from both a long-range coulombic attraction between the positively charged peptide and the negatively charged lipid bilayer, and a short-range Born or image charge repulsion, is a minimum when -2.5 A (i.e., one layer of water) exists between the van der Waals surfaces of the peptide and the lipid bilayer. The calculated molar association constants, K, agree well with the measured values: K is typically about 1 0-fold smaller than the experimental value (i.e., a difference of about 1.5 kcal/mol in the free energy of binding). The predicted dependence of K (or the binding free energies) on the ionic strength of the solution, the mol% of acidic lipids in the membrane, and the number of basic residues in the peptide agree very well with the experimental measurements. These calculations are relevant to the membrane binding of a number of important proteins that contain clusters of basic residues.
Applied Magnetic Resonance, 2005
Pulsed electron-electron double resonance (PELDOR) combined with continuous-wave electron paramagnetic resonance was used to study inter-and intramolecular dipole-dipole interactions between spin labels for spin-labeled analogs of trichogin GA IV bound to multilamellar membranes of egg L<t-phosphatidylcholine (ePC) and in ePC membranes containing cholesterol. AII samples were frozen to 77 K. For mono-labeled peptide concentrations in lipid over the range between 0.5 to 2.2 mol%, it is shown that in these membranes trichogin rnolecules are distributed homogeneously and are likely to be Iocated on or near the inner and outer membrane surfaces. Addition of cholesterol to a final concentration of 16.5 mol% leads to ah increase of the local concentration of trichogin molecules in the membranes. For the double-labeled trichogin, a distribution of the intramolecular distanee between the two spin labels was observed. The distribution function is characterized by two main maxima located at distances of 1.3 and 1.8 nm. The distance of 1.3 nm is close to that expected for the ct-helix structure of the peptide chain. The distance of 1.8 nm corresponds to a mixed structure in which a 3~0-helix is combined with a set of even more elongated conformations.
Biophysical Journal, 1998
Direct fluorescence digital imaging microscopy observations demonstrate that a basic peptide corresponding to the effector region of the myristoylated alanine-rich C kinase substrate (MARCKS) self-assembles into membrane domains enriched in the acidic phospholipids phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP 2). We show here that pentalysine, which corresponds to the first five residues of the MARCKS effector region peptide and binds to membranes through electrostatic interactions, also forms domains enriched in PS and PIP 2. We present a simple model of domain formation that represents the decrease in the free energy of the system as the sum of two contributions: the free energy of mixing of neutral and acidic lipids and the electrostatic free energy. The first contribution is always positive and opposes domain formation, whereas the second contribution may become negative and, at low ionic strength, overcome the first contribution. Our model, based on Gouy-Chapman-Stern theory, makes four predictions: 1) multivalent basic ligands, for which the membrane binding is a steep function of the mole fraction of acidic lipid, form domains enriched in acidic lipids; domains break up at high concentrations of either 2) basic ligand or 3) monovalent salt; and 4) if multivalent anionic lipids (e.g., PIP 2) are present in trace concentrations in the membrane, they partition strongly into the domains. These predictions agree qualitatively with experimental data obtained with pentalysine and spermine, another basic ligand.
Biochemistry, 1990
The interaction of interrelated model peptides with model membranes has been studied by techniques based on tryptophan fluorescence. The peptides used are derivatives of the sequence H-Ala-Met-Leu-Trp-Ala-OH, which wav designed for this purpose. Several modifications yielded a set of 13 pentaand hexapeptides varying in net charge, hydrophobicity, charge distribution, and the intramolecular position of the tryptophan residue with respect to the charge(s). The affinity of these peptides for small unilamellar vesicles (SUV) consisting of zwitterionic egg phosphatidylcholine (eggPC) and negatively charged beef heart cardiolipin (bhCL) has been investigated in a comparative way. The criteria for affinity comprise (1) intrinsic fluorescence changes upon titration of the peptides with the lipid vesicles, (2) reduced accessibility of the peptides to aqueous quenchers of tryptophan fluorescence (Iand acrylamide) in the presence of lipid, and (3) exposure to membrane-incorporated fluorescence quenchers, brominated phosphatidylcholines (BrPC). Application of BrPC brominated at different positions along the acyl chains provided information on the membrane topology of the peptides. With respect to the extent of affinity for zwitterionic membranes, the overall hydrophobicity of the peptides is the main determinant. A comparison of the affinity for PC of equally hydrophobic peptides carrying either a single positive or negative charge reveals preferential interaction of the cationic peptide. Both hydrophobic and electrostatic interactions determine the affinity of positively charged mono-and divalent peptides for C L vesicles. The distribution of the charged moieties in divalent positively charged peptides, either both at one end of the molecule or one at each end, has little influence on the affinity of these peptides for C L but does affect the extent of exposure to BrPC. Upon decreasing the surface charge density of the vesicles by diluting C L with increasing amounts of PC, both types of peptides show different behavior. The position of the tryptophan relative to the charged moiety in the peptide molecule is shown to affect the fluorescent properties upon interaction with vesicles. Concerning the membrane topology, all peptides adopt a localization near the membrane surface, with the neutral peptides inserting slightly deeper into the bilayer than the charged peptides. The results allow a comparative analysis of the factors determining the extents and modes of lipid-model peptide interaction; in addition, the validity of the methods applied is discussed. * To whom correspondence should be addressed. *Centre for Biomembranes and Lipid Enzymology. Institute of Molecular Biology and Medical Biotechnology. understanding of these mechanisms [for review see Jain and Zakim (1987)l. The synthetic peptide approach has the advantage of being flexible: the effects of small changes in the molecule on the lipid-peptide interaction can be determined. Several authors have studied large synthetic a-helix forming peptides which were shown to span the lipid bilayer, as a model for transmembrane proteins (Voges et al., 1987; Davis et al., 1988). Others have investigated much smaller (di-, tri-, tetra-, and penta-) peptides in interaction with model membranes. Jacobs and White (1986, 1987, 1989) have compared, from a thermodynamic point of view, the effect of single amino acid substitutions on the perturbation of DMPC' model membranes by tripeptides carrying a single positive charge (Ala-X-Ala-Abbreviations: DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DOPC, dioleoylphosphatidylcholine; DOPS, dioleoylphosphatidylserine; CL, cardiolipin from beef heart; eggPC, egg yolk phosphatidylcholine; BrPC, brominated phosphatidylcholine(s); 2-Br-PC, l-palmitoyl-2-(2-bromohexadecanoyl)phosphatidylcholine; 6,7-Br2-PC, l-palmitoyl-2-(6,7-dibromostearoyl)phosphatidylcholine; 9,10-Br2-PC, l-palmitoyl-2-(9, IO-dibromostearoy1)phosphatidylcholine; 1 1 ,12-Br2-PC, l-palmitoyl-2-(1 1,12-dibromostearoyl)phosphatidylcholine; 9,10-Br4-PC, 1,2-bis-(9,10-dibromostearoyl)phosphatidylcholine; t-Boc, terr-butyloxycarbonyl; TFA, trifluoroacetic acid; HPLC, high-performance liquid chromatography; SUV, small unilamellar vesicles; naf, normalized accessibility factor.
How lipids influence the mode of action of membrane-active peptides
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2007
The human, multifunctional peptide LL-37 causes membrane disruption by distinctly different mechanisms strongly dependent on the nature of the membrane lipid composition, varying not only with lipid headgroup charge but also with hydrocarbon chain length. Specifically, LL-37 induces a peptide-associated quasi-interdigitated phase in negatively charged phosphatidylglycerol (PG) model membranes, where the hydrocarbon chains are shielded from water by the peptide. In turn, LL-37 leads to a disintegration of the lamellar organization of zwitterionic dipalmitoyl-phosphatidylcholine (DPPC) into disk-like micelles. Interestingly, interdigitation was also observed for the longer-chain C18 and C20 PCs. This dual behavior of LL-37 can be attributed to a balance between electrostatic interactions reflected in different penetration depths of the peptide and hydrocarbon chain length. Thus, our observations indicate that there is a tight coupling between the peptide properties and those of the lipid bilayer, which needs to be considered in studies of lipid/peptide interaction. Very similar effects were also observed for melittin and the frog skin peptide PGLa. Therefore, we propose a phase diagram showing different lipid/peptide arrangements as a function of hydrocarbon chain length and LL-37 concentration and suggest that this phase diagram is generally applicable to membrane-active peptides localized parallel to the membrane surface.
Biochimica Et Biophysica Acta-general Subjects, 2008
Cell penetrating peptide Amphipathic peptide Peptide-membrane interaction Differential scanning calorimetry Circular dichroism 31 P NMR Independently from the cell penetrating peptide uptake mechanism (endocytic or not), the interaction of the peptide with the lipid bilayer remains a common issue that needs further investigation. The cell penetrating or antimicrobial properties of exogenous peptides require probably different preliminary interactions with the plasma membrane. Herein, we have employed 31 P NMR, differential scanning calorimetry and CD to study the membrane interaction and perturbation mechanisms of two basic peptides with similar length but distinct charge distribution, penetratin (non-amphipathic) and RL16, a secondary amphipathic peptide. The peptide effects on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dipalmitoleoyl phosphatidylethanolamine (DiPoPE) were investigated. We have found that, even though both peptides are cationic, their interaction with zwitterionic versus anionic lipids is markedly distinct. Penetratin greatly affects the temperature, enthalpy and cooperativity of DMPG main phase transition but does not affect those of DMPC while RL16 presents opposite effects. Additionally, it was found that penetratin induces a negative curvature whereas RL16 induces a positive one, since a decrease in the fluid lamellar to inverted hexagonal phase transition temperature of DiPoPE (T H ) was observed for penetratin and an increase for RL16. Contrary to penetratin, 31 P NMR of samples containing DMPC MLVs and RL16 shows an isotropic signal indicative of the formation of small vesicles, concomitant with a great decrease in sample turbidity both below and at the phase transition temperature. Opposite effects were also observed on DMPG where both peptides provoke strong aggregation and precipitation. Both CPPs adopt helical structures when contacting with anionic lipids, and possess a dual behavior by either presenting their cationic or hydrophobic domains towards the phospholipid face, depending on the lipid nature (anionic vs zwitterionic, respectively). Surprisingly, the increase of electrostatic interactions at the water membrane interface prevents the insertion of RL16 hydrophobic region in the bilayer, but is essential for the interaction of penetratin. Modulation of amphipathic profiles and charge distribution of CPPs can alter the balance of hydrophobic and electrostatic membrane interaction leading to translocation or and membrane permeabilisation. Penetratin has a relative pure CPP behavior whereas RL16 presents mixed CPP/AMP properties. A better understanding of those processes is essential to unveil their cell translocation mechanism.
The journal of physical chemistry. B, 2014
We investigated the dependence of membrane binding on amino acid sequence for a series of amphipathic peptides derived from δ-lysin. δ-Lysin is a 26 amino acid, N-terminally formylated, hemolytic peptide that forms an amphipathic α-helix bound at membrane-water interfaces. A shortened peptide, lysette, was derived from δ-lysin by deletion of the four N-terminal amino acid residues. Five variants of lysette were synthesized by altering the amino acid sequence such that the overall hydrophobic moment remained essentially the same for all peptides. Peptide-lipid equilibrium dissociation constants and helicities of peptides bound to zwitterionic lipid vesicles were determined by stopped-flow fluorescence and circular dichroism. We found that binding to phosphatidylcholine bilayers was a function of the helicity of the bound peptide alone and independent of the a priori hydrophobic moment or the ability to form intramolecular salt bridges. Molecular dynamics (MD) simulations on two of th...