The interaction of neuropeptide Y with negatively charged and zwitterionic phospholipid membranes.

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 alpha-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 from (1)H magic-angle spinning NMR relaxation measurements using spin-labelled [Ala(31), TOAC(32)]-NPY. The results suggest that the immersion depth of NPY into the membrane is triggered by the membrane composition. The alpha-helix of NPY is located in the upper chain region of zwitterionic membranes but its position is shifted to the glycerol region in negatively charged membranes. For membranes composed of phosphatidylcholine and phosphatidylserine, an intermediate position of the alpha-helix is observed.

Annexin A2/P11 interaction: new insights into annexin A2 tetramer structure by chemical crosslinking, high-resolution mass spectrometry, and computational modeling.

During the past few years, the structural analysis of proteins and protein complexes by chemical crosslinking and mass spectrometry has enjoyed increasing popularity. With this approach we have investigated the quaternary structure of the complex between annexin A2 and p11, which is involved in numerous cellular processes. Although high-resolution data are available for both interaction partners as well as for the complex between two p11 subunits and two annexin A2 N-terminal peptides, the structure of the complete annexin A2/p11 heterotetramer has not yet been solved at high resolution. Thus, the quaternary structure of the biologically relevant, membrane-bound annexin A2/p11 complex is still under discussion, while the existence of a heterotetramer or a heterooctamer is the prevailing opinion. We gained further insight into the spatial organization of the annexin A2/p11 heterotetramer by employing chemical crosslinking combined with high-resolution mass spectrometry. Furthermore, tandem mass spectrometry served as a tool for an exact localization of crosslinked amino acid residues and for a confirmation of crosslinked product assignment. On the basis of distance constraints from the crosslinking data we derived structural models of the annexin A2/p11 heterotetramer by computational docking with Rosetta. We propose an octameric model for the annexin A2/p11 complex, which exerts annexin A2 function. The proposed structure of the annexin A2/p11 octamer differs from so far suggested models and sheds new light into annexin A2/p11 interaction.

Annexin A4 binding to anionic phospholipid vesicles modulated by pH and calcium.

Annexin A4 belongs to a class of Ca(2+)-binding proteins for which different functions in the cell have proposed, e.g. involvement in exocytosis and in the coagulation process. All these functions are related to the ability of the annexins to bind to acidic phospholipids. In this study the interaction of annexin A4 with large unilamellar vesicles (LUV) prepared from phosphatidylserine (PS) or from phosphatidic acid (PA) is investigated at neutral and acidic pH. Annexin A4 strongly binds to either lipid at acidic pH, whereas at neutral pH only weak binding to PA and no binding to PS occurs. Addition of 40 microM Ca(2+) leads to a strong binding to the lipids also at neutral pH. This is caused by the different electric charge of the protein below and above its isoelectric point. Binding of annexin A4 induces dehydration of the vesicle surface. The strength of the effects is much greater at pH 4 than at pH 7.4. At pH 7.4 annexin A4 reduces the Ca(2+)-threshold concentration necessary to induce fusion of PA LUV. The Ca(2+) induced fusion of PS LUV is not affected by annexin A4 at pH 7.4. At pH 4 annexin A4 induces fusion of either vesicles without Ca(2+). Despite the low binding extents at neutral pH annexin A4 induces a Ca(2+) independent leakage of PS- or PA-LUV. The leakage extent is increased at acidic pH. From the data two suggestions are made: (1) At pH 4 annexin A4 (at least partially) penetrates into the bilayer in contrast to the preferred location at the vesicle surface at neutral pH. The conformation of annexin A4 seems to be different at the two conditions. (2) At neutral pH, Annexin A4 seems to be able to bind two PA vesicles simultaneously; however, only one PS vesicle at the same time. This behavior might be related to a recently described double Ca(2+) binding site, which appears to be uniquely suited for PS.

The suitability of different DHB isomers as matrices for the MALDI-TOF MS analysis of phospholipids: which isomer for what purpose?

Although the analysis of large biomolecules is the prime application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), there is also increasing interest in lipid analysis. Since lipids possess relatively small molecular weights, matrix signals should be as small as possible to avoid overlap with lipid peaks. Although 2,5-dihydroxybenzoic acid (DHB) is an established MALDI matrix, the question whether just this isomer is ideal for lipid analysis was not yet addressed. UV absorptions of all six DHB isomers were determined and their laser desorption spectra recorded. In addition, all isomers were used as matrices to record positive and negative ion mass spectra of selected phospholipids (phosphatidylcholine and -serine): In the order 2,5-, 2,6-, 2,3- and 2,4-DHB, the quality of the positive ion lipid spectra decreases. This correlates well with the decreasing acidity of the applied DHB isomers. The 3,4- and 3,5- isomers give only very weak positive ion signals especially of acidic lipids. In contrast, the most suitable matrices in the negative ion mode are 2,5-, 2,4- and 3,5-DHB. 2,6-DHB does not provide any signal in the negative ion mode due to its marked acidity. Finally, differences in the crystallization behavior of the pure matrix and the matrix/lipid co-crystals were also monitored by atomic force microscopy (AFM): 2,5-DHB gave the smallest crystals and the skinniest layer. It is concluded that basically all DHB isomers can be used as MALDI matrices but the 2,5-isomer represents the most versatile compound.

MALDI-TOF MS in lipidomics.

So far, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) seemed to be nearly a synonym for protein analysis. However, there is growing evidence that this technique is also an useful tool in lipid analysis and lipidomics because of its fast, simple and convenient performance allowing to record mass spectra of cells, crude tissue or body fluid extracts or even intact tissue slices in a few minutes. On the negative side, however, the reproducibility of MALDI-TOF mass spectra depends significantly on the homogeneity of the co-crystals between matrix and analyte and different lipid classes are detected with different sensitivities. This is especially important because lipids with quaternary ammonia groups (e.g., GPCho) may prevent the detection of other lipid classes (e.g., GPEtn). This review starts with a short overview on traditional methods of lipid analysis with the focus on mass spectrometric methods and compares MALDI-TOF MS with other important ionization techniques. Afterwards, some landmarks in the development of MALDI-TOF MS will be introduced and some important examples in the field of tissue and body fluid lipid analysis will be discussed. This review ends with a short outlook and summary focusing on the advantages and drawbacks of MALDI-TOF MS in lipidomics.

Membrane binding and structure of de novo designed alpha-helical cationic coiled-coil-forming peptides.

We introduce a de novo designed peptide model system that enables the systematic study of 1) the role of a membrane environment in coiled-coil peptide folding, 2) the impact of different domains of an alpha-helical coiled-coil heptad repeat on the interaction with membranes, and 3) the dynamics of coiled-coil peptide-membrane interactions depending on environmental conditions. Starting from an ideal alpha-helical coiled-coil peptide sequence, several positively charged analogues were designed that exhibit a high propensity toward negatively charged lipid membranes. Furthermore, these peptides differ in their ability to form a stable alpha-helical coiled-coil structure. The influence of a membrane environment on peptide folding is studied. All positively charged peptides show strong interactions with negatively charged membranes. This interaction induces an alpha-helical structure of the former random-coil peptides, as revealed by circular dichroism measurements. Furthermore, vesicle aggregation is induced by a coiled-coil interaction of vesicle-bound peptides. Dynamic light scattering experiments show that the strength of vesicle aggregation increases with the peptide's intrinsic ability to form a stable alpha-helical coiled coil. Thus, the peptide variant equipped with the strongest inter- and intra-helical coiled-coil interactions shows the strongest effect on vesicle aggregation. The secondary structure of this peptide in the membrane-bound state was studied as well as its effect on the phospholipids. Peptide conformation within the peptide-lipid aggregates was analyzed by (13)C cross-polarization magic-angle spinning NMR experiments. A uniformly (13)C- and (15)N-labeled Leu residue was introduced at position 12 of the peptide chain. The (13)C chemical shift and torsion angle measurements support the finding of an alpha-helical structure of the peptide in its membrane-bound state. Neither membrane leakage nor fusion was observed upon peptide binding, which is unusual for amphiphatic peptide structures. Our results lay the foundation for a systematic study of the influence of the alpha-helical coiled-coil folding motif in membrane-active events on a molecular level.

Structural determinants for plant annexin-membrane interactions.

The interactions of two plant annexins, annexin 24(Ca32) from Capsicum annuum and annexin Gh1 from Gossypium hirsutum, with phospholipid membranes have been characterized using liposome-based assays and adsorption to monolayers. These two plant annexins show a preference for phosphatidylserine-containing membranes and display a membrane binding behavior with a half-maximum calcium concentration in the sub-millimolar range. Surprisingly, the two plant annexins also display calcium-independent membrane binding at levels of 10-20% at neutral pH. This binding is regulated by three conserved surface-exposed residues on the convex side of the proteins that play a pivotal role in membrane binding. Due to quantitative differences in the membrane binding behavior of N-terminally His-tagged and wild-type annexin 24(Ca32), we conclude that the N-terminal domain of plant annexins plays an important role, reminiscent of the findings in their mammalian counterparts. Experiments elucidating plant annexin-mediated membrane aggregation and fusion, as well as the effect of these proteins on membrane surface hydrophobicity, agree with findings from the membrane binding experiments. Results from electron microscopy reveal elongated rodlike assemblies of plant annexins in the membrane-bound state. It is possible that these structures consist of protein molecules directly interacting with the membrane surface and molecules that are membrane-associated but not in direct contact with the phospholipids. The rodlike structures would also agree with the complex data from intrinsic protein fluorescence. The tubular lipid extensions suggest a role in the membrane cytoskeleton scaffolding or exocytotic processes. Overall, this study demonstrates the importance of subtle changes in an otherwise conserved annexin fold where these two plant annexins possess distinct modalities compared to mammalian and other nonplant annexins.

Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases.

Elevated low-density lipoprotein (LDL) levels induce activation of the p38 mitogen-activated protein kinase (MAPK), a stress-activated protein kinase potentially participating in the development of atherosclerosis. The nature of the lipoprotein components inducing p38 MAPK activation has remained unclear however. We show here that both LDLs and high-density lipoproteins (HDLs) have the ability to stimulate the p38 MAPKs with potencies that correlate with their cholesterol content. Cholesterol solubilized in methyl-beta-cyclodextrin was sufficient to activate the p38 MAPK pathway. Liposomes made of phosphatidylcholine (PC) or sphingomyelin, the two main phospholipids found in lipoproteins, were unable to stimulate the p38 MAPKs. In contrast, PC liposomes loaded with cholesterol potently activated this pathway. Reducing the cholesterol content of LDL particles lowered their ability to activate the p38 MAPKs. Cell lines representative of the three main cell types found in blood vessels (endothelial cells, smooth muscle cells and fibroblasts) all activated their p38 MAPK pathway in response to LDLs or cholesterol-loaded PC liposomes. These results indicate that elevated cholesterol content in lipoproteins, as seen in hypercholesterolemia, favors the activation of the stress-activated p38 MAPK pathway in cells from the vessel wall, an event that might contribute to the development of atherosclerosis.

Membrane interaction of neuropeptide Y detected by EPR and NMR spectroscopy.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals. It belongs to the best-conserved peptides in nature, i.e., the amino acid sequences of even evolutionary widely separated species are very similar to each other. Using porcine NPY, which differs from human NPY only at position 17 (a leucine residue exchanged for a methionine), labeled with a TOAC spin probe at the 2nd, 32nd, or 34th positions of the peptide backbone, the membrane binding and penetration of NPY was determined using EPR and NMR spectroscopy. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the analysis of the EPR line shapes, the spectral contributions of free, dimerized, and membrane bound NPY could be separated. This analysis was further supported by quenching experiments, which selected the contributions of the bound NPY fraction. The results of this study give rise to a model where the alpha-helical part of NPY (amino acids 13-36) penetrates the membrane interface. The unstructured N-terminal part (amino acids 1-12) extends into the aqueous phase with occasional contacts with the lipid headgroup region. Besides the mixing ratio of zwitterionic and negatively charged phospholipid species, the electrostatic peptide membrane interactions are influenced by the pH value, which determines the net charge of the peptide resulting in a modified membrane binding affinity. The results of these variations indicate that NPY binding to phospholipid membranes depends strongly on the electrostatic interactions. An estimation of the transfer energy of the peptide from aqueous solution to the membrane interface DeltaG supports the preferential interaction of NPY with negatively charged membranes.

Modulation of lysozyme charge influences interaction with phospholipid vesicles.

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between charge state of the protein and its interaction with negatively charged phospholipid membranes chemical modifications of the proteins were carried out. Succinylation and carbodiimide modification was used to shift the isoelectric point of lysozyme to lower and higher pH values, respectively. The binding of the modified lysozyme to phospholipid vesicles prepared from phosphatidic acid (PA) was determined using microelectrophoresis and ultracentrifugation. At acidic pH of the solution all lysozyme species reduced the surface charges of PA vesicles. Succinylated lysozyme (succ lysozyme) reduced the electrophoretic mobility (EPM) to nearly zero, whereas native lysozyme and carboxylated lysozyme (carbo lysozyme) changed the surface charge to positive values. At neutral pH, the reduction of surface charges was less for carbo lysozyme and unmodified lysozyme. Succ lysozyme did not change the EPM. Unmodified and carbo lysozyme decreased the magnitude of EPM, but the whole complex was still negatively charged. The bound fraction of all modified lysozyme to PA vesicles at high lysozyme/PA ratios was nearly constant at acidic pH. At low lysozyme/PA ratios the extent of bound lysozyme is changed in the order carbo>unmodified>succ lysozyme. Increasing the pH, the extent of bound lysozyme to PA large unilamellar vesicles (LUV) is reduced, at pH 9.0 only 35% of carbo lysozyme, 23% of unmodified lysozyme is bound, whereas succ lysozyme does not bind at pH 7.4 and 9.0. At low pH, addition of all lysozyme species resulted in a massive aggregation of PA liposomes, at neutral pH aggregation occurs at much higher lysozyme/PA ratios. Lysozyme binding to PA vesicles is accompanied by the penetration of lysozyme into the phospholipid membrane as measured by monolayer techniques. The penetration of lysozyme into the monolayer was modulated by pH and ionic strengths. The interaction of lysozyme with negatively charged vesicles leads to a decrease of the phospholipid vesicle surface hydration as measured by the shift of the maximum of the fluorescence signal of a headgroup labeled phospholipid. The binding of bis-ANS as an additional indicator for the change of surface hydrophobicity is increased at low pH after addition of lysozyme to the vesicles. More hydrophobic patches of the lysozyme-PA complex are exposed at low pH. At low pH the binding process of lysozyme to PA vesicles is followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the vesicle aqueous content. The extent of lysozyme interaction with PA LUV at neutral and acidic pH is in the order carbo lysozyme>lysozyme>succ lysozyme.

Cholesterol esterase action on human high density lipoproteins and inhibition studies: detection by MALDI-TOF MS.

The modification of lipoproteins by lipolytic enzymes such as cholesterol esterase (CEase) is assumed to play an important role in the pathogenesis of atherosclerosis. However, details of the activation and inhibition of CEase are still unknown. In this study, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to investigate the extracts of human lipoproteins after treatment with CEase and to monitor the effects of the inhibitor 2-(diethylamino)-6,7-dihydro-4H,5H-cyclopenta[4,5] thieno[2,3-d][1,3]oxazin-4-one (DOT-3). This approach has the advantage that all lipid classes can be independently detected; therefore, conclusions on the mechanism of the applied enzyme are possible. Besides the expected decrease of cholesteryl esters (CEs) in HDL, a significantly enhanced content of lysophosphatidylcholine (LPC) was also detected, confirming the broad substrate specificity of CEase. It was also demonstrated that DOT-3 significantly inhibited the CEase-catalyzed cleavage of CEs in HDL. Phospholipid (PL) vesicles prepared from phosphatidylcholine (PC) or PC and cholesteryl linoleate were treated with CEase, and the changes in lipid composition were investigated. From the analysis of the generated LPC species in HDL and in the isolated lipid mixtures, it is evident that CEase catalyzes the cleavage of the fatty acid residues in both the sn-1 and sn-2 positions of the PLs. These effects are obvious in the absence as well as in the presence of detergents.

Effective dephosphorylation of Src substrates by SHP-1.

The protein-tyrosine phosphatase SHP-1 is a negative regulator of multiple signal transduction pathways. We observed that SHP-1 effectively antagonized Src-dependent phosphorylations in HEK293 cells. This occurred by dephosphorylation of Src substrates, because Src activity was unaffected in the presence of SHP-1. One reason for efficient dephosphorylation was activation of SHP-1 by Src. Recombinant SHP-1 had elevated activity subsequent to phosphorylation by Src in vitro, and SHP-1 variants with mutated phosphorylation sites in the C terminus, SHP-1 Y538F, and SHP-1 Y538F,Y566F were less active toward Src-generated phosphoproteins in intact cells. A second reason for efficient dephosphorylation is the substrate selectivity of SHP-1. Pull-down experiments with different GST-SHP-1 fusion proteins revealed efficient interaction of Src-generated phosphoproteins with the SHP-1 catalytic domain rather than with the SH2 domains. Phosphopeptides that correspond to good Src substrates were efficiently dephosphorylated by SHP-1 in vitro. Phosphorylated "optimal Src substrate" AEEEIpYGEFEA (where pY is phosphotyrosine) and a phosphopeptide corresponding to a recently identified Src phosphorylation site in p120 catenin, DDLDpY(296)GMMSD, were excellent SHP-1 substrates. Docking of these phosphopeptides into the catalytic domain of SHP-1 by molecular modeling was consistent with the biochemical data and explains the efficient interaction. Acidic residues N-terminal of the phosphotyrosine seem to be of major importance for efficient substrate interaction. Residues C-terminal of the phosphotyrosine probably contribute to the substrate selectivity of SHP-1. We propose that activation of SHP-1 by Src and complementary substrate specificities of SHP-1 and Src may lead to very transient Src signals in the presence of SHP-1.

Evaluation of the metal binding properties of a histidine-rich fusogenic peptide by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) was used to investigate metal ion interactions of the 18 amino acid peptide fragment B18 (LGLLLRHLRHHSNLLANI), derived from the membrane-associated protein bindin. The peptide sequence B18 represents the minimal membrane-binding motif of bindin and resembles a putative fusion peptide. The histidine-rich peptide has been shown to self-associate into distinct supramolecular structures, depending on the presence of Zn(2+) and Cu(2+). We examined the binding of B18 to the metal ions Cu(2+), Zn(2+), Mg(2+), Ca(2+), Mn(2+) and La(3+). For Cu(2+), we compared the metal binding affinities of the wild-type B18 peptide with those of its mutants in which one, two or three histidine residues have been replaced by serines. Upon titration of B18 with Cu(2+) ions, we found sequential binding of two Cu(2+) ions with dissociation constants of approximately 34 and approximately 725 micro M. Mutants of B18, in which one histidine residue is replaced by serine, still exhibit sequential binding of two copper ions with affinities for the first Cu(2+) ion comparable to that of wild-type B18 peptide, but with a greatly reduced affinity for the second Cu(2+) ion in mutants H112S and H113S. For mutants in which two histidines are replaced by serines, the affinity for the first Cu(2+) ion is reduced approximately 3-10 times in comparison with B18. The mutant in which all three histidine residues are replaced by serines exhibits an approximately 14-fold lower binding for the first Cu(2+) ion compared with B18. For the other metal ions under investigation (Zn(2+), Mg(2+), Ca(2+), Mn(2+) and La(3+)), a modest affinity to B18 was detected binding to the peptide in a 1 : 1 stoichiometry. Our results show a high affinity of the wild-type fusogenic peptide B18 for Cu(2+) ions whereas the Zn(2+) affinity was found to be comparable to that of other di- and trivalent metal ions.

Increased non-viral gene transfer levels in mice by concentration of cationic lipid DNA complexes formed under optimized conditions.

BACKGROUND: A number of non-viral gene transfer reagents including cationic lipid DNA complexes (LDC) have been developed and were improved by changing the ratio of their components. To determine the effect of other parameters during complex formation affecting the efficacy of LDC, the conditions during complexation were varied without changing the ratios of the components. METHODS: LDC were formed at fixed ratios of an equimolar mixture of DOTAP and cholesterol to DNA to the condensing agent protamine sulfate according to different protocols at varying final concentrations. The influence of these parameters on transfection efficiency and physical properties of the complexes was determined. RESULTS: Changing the order of addition of compounds during complex formation affected the size distribution, the charge of the LDC, the interaction between the lipids and the accessibility of the DNA. At fixed ratios of the components, higher transfection efficiencies were observed with more condensed LDC. Complexation in higher volumes increased transduction efficiency of the complexes after intravenous inoculation. Due to restrictions on the injectable volume, the LDC were formed in the optimal volume and subsequently concentrated by ultrafiltration. The concentrated complexes maintained transduction efficiency and up to 60-fold higher in vivo transduction levels were obtained. CONCLUSIONS: In addition to the ratio of the components of cationic lipid DNA complexes, the final concentration and the order of addition of compounds during complex formation are critical for high transduction efficiency. Concentration of LDC formed under optimal conditions can be used to further increase in vivo gene transfer levels.

Structural and dynamical changes of the bindin B18 peptide upon binding to lipid membranes. A solid-state NMR study.

Structural and dynamical features of the B18 peptide from the sea urchin sperm binding protein were determined in the crystalline state and in zwitterionic lipid bilayers at a peptide:lipid molar ratio of 1:12 using solid-state NMR spectroscopy. The study was focused on three (13)C and (15)N uniformly labeled leucine residues, which were introduced into three different B18 peptides at positions evenly distributed along the B18 primary structure. Isotropic (13)C and (15)N chemical shift measurements showed that while B18 possesses a nonhelical and non-sheet-like structure in the crystalline state, the peptide adopts an oligomeric beta-sheet structure in the membrane in the presence of Zn(2+) ions at high peptide:lipid ratio. Torsion angle measurements for the three leucine sites supported these results, with phi torsion angles between -80 degrees and -90 degrees in the crystalline state and between -110 degrees and -120 degrees in the membrane-bound form. These phi torsion angles determined for membrane-bound B18 are consistent with a parallel beta-sheet secondary structure. Analysis of motionally averaged dipolar coupling measurements established an increase of the mobility in the leucine side chains upon binding to the membrane, whereas the backbone mobility remained essentially unchanged, except in the binding site of Zn(2+) ions. This difference in mobility was related to the H-bond network in the parallel beta-sheet structure, which involves the backbone and excludes the side chains of leucine residues. The parallel beta-sheet structure of B18 in the membrane in the presence of Zn(2+) appears to be an active state for the fusion of zwitterionic membranes in the presence of Zn(2+). A fluorescence fusion assay indicated that high B18 concentrations are required to induce fusion in these systems. Therefore, it was hypothesized that the oligomeric beta-sheet secondary structure revealed in the study represents an active state of the peptide in a membrane environment during fusion.

Membrane insertion of a lipidated ras peptide studied by FTIR, solid-state NMR, and neutron diffraction spectroscopy.

Membrane binding of a doubly lipid modified heptapeptide from the C-terminus of the human N-ras protein was studied by Fourier transform infrared, solid-state NMR, and neutron diffraction spectroscopy. The 16:0 peptide chains insert well into the 1,2-dimyristoyl-sn-glycero-3-phosphocholine phospholipid matrix. This is indicated by a common main phase transition temperature of 21.5 degrees C for both the lipid and peptide chains as revealed by FTIR measurements. Further, (2)H NMR reveals that peptide and lipid chains have approximately the same chain length in the liquid crystalline state. This is achieved by a much lower order parameter of the 16:0 peptide chains compared to the 14:0 phospholipid chains. Finally, proton/deuterium contrast variation of neutron diffraction experiments indicates that peptide chains are localized in the membrane interior analogous to the phospholipid chains. In agreement with this model of peptide chain insertion, the peptide part is localized at the lipid-water interface of the membrane. This is revealed by (1)H nuclear Overhauser enhancement spectra recorded under magic angle spinning conditions. Quantitative cross-peak analysis allows the examination of the average location of the peptide backbone and side chains with respect to the membrane. While the backbone shows the strongest cross-relaxation rates with the phospholipid glycerol, the hydrophobic side chains of the peptide insert deeper into the membrane interior. This is supported by neutron diffraction experiments that reveal a peptide distribution in the lipid-water interface of the membrane. Concurring with these experimental findings, the amide protons of the peptide show strong water exchange as seen in NMR and FTIR measurements. No indications for a hydrogen-bonded secondary structure of the peptide backbone are found. Therefore, membrane binding of the C-terminus of the N-ras protein is mainly due to lipid chain insertion but also supported by interactions between hydrophobic side chains and the lipid membrane. The peptide assumes a mobile and disordered conformation in the membrane. Since the C-terminus of the soluble part of the ras protein is also disordered, we hypothesize that our model for membrane binding of the ras peptide realistically describes the membrane binding of the lipidated C-terminus of the active ras protein.