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BP100 is a cationic undecamer peptide with antimicrobial and cell-penetrating activities. The orientation of this amphiphilic α-helix in lipid bilayers was examined under numerous conditions using solid-state 19 F, 15 N and 2 H NMR. At high temperatures in saturated phosphatidylcholine lipids, BP100 lies flat on the membrane surface, as expected. Upon lowering the temperature towards the lipid phase transition, the helix is found to flip into an upright transmembrane orientation. In thin bilayers, this inserted state was stable at low peptide concentration, but thicker membranes required higher peptide concentrations. In the presence of lysolipids, the inserted state prevailed even at high temperature. Molecular dynamics simulations suggest that BP100 monomer insertion can be stabilized by snorkeling lysine side chains. These results demonstrate that even a very short helix like BP100 can span (and thereby penetrate through) a cellular membrane under suitable conditions., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

In this study, we investigate how the length of amphiphilic β-sheet forming peptides affects their interaction with membranes. Four polycationic model peptides with lengths from 6 to 18 amino acids were constructed from simple Lys-Leu repeats, giving [KL] n=3,5,7,9 . We found that (1) they exhibit a pronounced antimicrobial activity with an intriguing length dependent maximum for [KL] 5 with 10 amino acids; (2) their hemolytic effect, on the other hand, increases steadily with peptide length. CD analysis (3) and TEM (4) show that all peptides-except for the short [KL] 3 -aggregate into amyloid-like fibrils in the presence of phosphate ions, which in turn has a critical effect on the results in (1) and (2). In fact, (5) vesicle leakage reveals an intrinsic membrane-perturbing activity (at constant peptide mass) of [KL] 5  > [KL] 9  > [KL] 7 in phosphate buffer, which changes to [KL] 5  ≈ [KL] 7  ≈ [KL] 9 in PIPES. A specific interaction with phosphate ions thus explains the subtle balance between two counteracting effects: phosphate-induced unproductive pre-aggregation in solution versus monomeric membrane binding and vigorous lipid perturbation due to self-assembly of the bound peptides within the bilayer. This knowledge can now be used to control and optimize the peptides in further applications.

The membrane alignment of helical amphiphilic peptides in oriented phospholipid bilayers can be obtained as ensemble and time averages from solid state 2 H NMR by fitting the quadrupolar splittings to ideal α-helices. At the same time, molecular dynamics (MD) simulations can provide atomistic insight into peptide-membrane systems. Here, we evaluate the potential of MD simulations to complement the experimental NMR data that is available on three exemplary systems: the natural antimicrobial peptide PGLa and the two designer-made peptides MSI-103 and KIA14, whose sequences were derived from PGLa. Each peptide was simulated for 1 μs in a DMPC lipid bilayer. We calculated from the MD simulations the local angles which define the side chain geometry with respect to the peptide helix. The peptide orientation was then calculated (i) directly from the simulation, (ii) from back-calculated MD-derived NMR splittings, and (iii) from experimental 2 H NMR splittings. Our findings are that (1) the membrane orientation and secondary structure of the peptides found in the NMR analysis are generally well reproduced by the simulations; (2) the geometry of the side chains with respect to the helix backbone can deviate significantly from the ideal structure depending on the specific residue, but on average all side chains have the same orientation; and (3) for all of our peptides, the azimuthal rotation angle found from the MD-derived splittings is about 15° smaller than the experimental value.

The amphipathic α-helical peptide KIA14 [(KIAGKIA) 2 -NH 2 ] was studied in membranes using circular dichroism and solid-state NMR spectroscopy to obtain global as well as local structural information. By analyzing 2 H NMR data from 10 analogues of KIA14 that were selectively labeled with Ala- d 3 , those positions that are properly folded into a helix could be determined within the membrane-bound peptide. The N-terminus was found to be unraveled, whereas positions 4-14 formed an ideal helix all the way to the C-terminus. The helicity did not change when Gly residues were replaced by Ala- d 3 but was reduced when Ile was replaced, indicating that large hydrophobic residues are required for membrane binding and helix formation. The reduced helicity was strongly correlated with a decrease in peptide-induced leakage from lipid vesicles. The orientation of the short KIA14 peptide was assessed in several lipid systems and compared with that of the longer KIA21 sequence [(KIAGKIA) 3 -NH 2 ]. In 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine, both peptides are aligned flat on the membrane surface, whereas in 1,2-dimyristoyl- sn-glycero-3-phosphatidylcholine (DMPC)/1-myristoyl-2-hydroxy- sn-glycero-3-phosphatidylcholine (lyso-MPC) both are inserted into the membrane in an upright orientation. These two types of lipid systems had been selected for their strongly negative and positive spontaneous curvature, respectively. We propose that in these cases, the peptide orientation is largely determined by the lipid properties. On the other hand, in plain DMPC and 1,2-dilauroyl- sn-glycero-3-phosphatidylcholine, which have only a slight positive curvature, a marked difference in orientation is evident: the short KIA14 lies almost flat on the membrane surface, whereas the longer KIA21 is more tilted. We thus propose that out of the lipid systems tested here, DMPC (with hardly any curvature) is the least biased lipid system in which peptide orientation and realignment can be studied, allowing to compare and discriminate the intrinsic effects of the properties of the peptides as such.

Choline-binding repeats (CBRs) are ubiquitous sequences with a β-hairpin core that are found in the surface proteins of several microorganisms such as S. pneumoniae (pneumococcus). Previous studies on a 14-mer CBR sequence derived from the pneumoccal LytA autolysin (LytA 239-252 peptide) have demonstrated a switch behaviour for this peptide, so that it acquires a stable, native-like β-hairpin conformation in aqueous solution but is reversibly transformed into an amphipathic α-helix in the presence of detergent micelles. With the aim of understanding the factors responsible for this unusual β-hairpin to α-helix transition, and to specifically assess the role of peptide hydrophobicity and helical amphipathicity in the process, we designed a series of LytA 239-252 variants affecting these two parameters and studied their interaction with dodecylphosphocholine (DPC) micelles by solution NMR, circular dichroism and fluorescence spectroscopies. Our results indicate that stabilising cross-strand interactions become essential for β-hairpin stability in the absence of optimal turn sequences. Moreover, both amphipathicity and hydrophobicity display comparable importance for helix stabilisation of CBR-derived peptides in micelles, indicating that these sequences represent a novel class of micelle/membrane-interacting peptides., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

SSL-25 (SSLLEKGLDGAKKAVGGLGKLGKDA) is one of the shortest peptides present in human sweat and is produced after the proteolytic processing of the parent peptide dermcidin. Both peptides are reported to have antimicrobial function. To determine the structure of SSL-25 in lipid bilayers, a series of 19 F-labeled SSL-25 analogs were synthesized. Circular dichroism (CD) analysis showed that SSL-25 and all of its analogs formed α-helices in the presence of lipid vesicles, thus allowing a detailed analysis via oriented CD and solid-state NMR. The results suggest that SSL-25 resides on the membrane surface with a slight helix tilt angle. A detailed 19 F NMR analysis revealed that SSL-25 does not form a continuous helix. The α-helical structure of the N-terminal part of the peptide was preserved in membranes of different lipid compositions and at various peptide-to-lipid molar ratios, but the C-terminus was disordered and did not fold into a well-defined α-helical conformation. Furthermore, the NMR results showed that SSL-25 resides on the membrane surface and does not re-orient into the membrane in response to changes in either peptide concentration or membrane composition. SSL-25 does not aggregate and remains fully mobile within the membrane bilayer, as shown by 19 F NMR. SSL-25 has a high binding affinity toward bilayers mimicking bacterial lipid compositions, but does not bind to mammalian model membranes containing cholesterol. These observations may explain the selectivity of this peptide for bacterial membranes, and they are also in line with basic biophysical considerations on spontaneous lipid curvature and the general effect of cholesterol on peptide/lipid interactions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A series of nine amphiphilic, pore-forming α-helical KIA peptides (KIAGKIA repeats) with lengths between 14 and 28 residues were studied by solid-state (15)N NMR to determine their alignment in oriented lipid bilayers. In a 2:1 mixture of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) with its corresponding 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-MPC), which has a highly positive spontaneous curvature, the helix tilt angle was found to vary steadily with peptide length. The shortest peptide was aligned transmembrane and upright, while the longer ones successively became tilted away from the membrane normal. This behavior is in agreement with the hydrophobic matching concept, conceived so far only for hydrophobic helices. In 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, with a negative spontaneous curvature, all KIA peptides remained flat on the bilayer surface, while the cylindrical DMPC lipids permitted a slight tilt. Peptide insertion thus depends critically on the intrinsic lipid curvature, and helix orientation is then fine-tuned by membrane thickness. A refined toroidal pore model is proposed.

Choline-binding modules (CBMs) have a ββ-solenoid structure composed of choline-binding repeats (CBR), which consist of a β-hairpin followed by a short linker. To find minimal peptides that are able to maintain the CBR native structure and to evaluate their remaining choline-binding ability, we have analysed the third β-hairpin of the CBM from the pneumococcal LytA autolysin. Circular dichroism and NMR data reveal that this peptide forms a highly stable native-like β-hairpin both in aqueous solution and in the presence of trifluoroethanol, but, strikingly, the peptide structure is a stable amphipathic α-helix in both zwitterionic (dodecylphosphocholine) and anionic (sodium dodecylsulfate) detergent micelles, as well as in small unilamellar vesicles. This β-hairpin to α-helix conversion is reversible. Given that the β-hairpin and α-helix differ greatly in the distribution of hydrophobic and hydrophilic side chains, we propose that the amphipathicity is a requirement for a peptide structure to interact and to be stable in micelles or lipid vesicles. To our knowledge, this "chameleonic" behaviour is the only described case of a micelle-induced structural transition between two ordered peptide structures., (© 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.)

Hydrophobic mismatch is a well-recognized principle in the interaction of transmembrane proteins with lipid bilayers. This concept was extended here to amphipathic membranolytic α-helices. Nine peptides with lengths between 14 and 28 amino acids were designed from repeated KIAGKIA motifs, and their helical nature was confirmed by circular dichroism spectroscopy. Biological assays for antimicrobial activity and hemolysis, as well as fluorescence vesicle leakage and solid-state NMR spectroscopy, were used to correlate peptide length with membranolytic activity. These data show that the formation of transmembrane pores is only possible under the condition of hydrophobic matching: the peptides have to be long enough to span the hydrophobic bilayer core to be able to induce vesicle leakage, kill bacteria, and cause hemolysis. By correlating the threshold lengths for biological activity with the biophysical results on model vesicles, the peptides could be utilized as molecular rulers to measure the membrane thickness in different cells.

The interaction of membranes with peptides and proteins is largely determined by their amphiphilic character. Hydrophobic moments of helical segments are commonly derived from their two-dimensional helical wheel projections, and the same is true for β-sheets. However, to the best of our knowledge, there exists no method to describe structures in three dimensions or molecules with irregular shape. Here, we define the hydrophobic moment of a molecule as a vector in three dimensions by evaluating the surface distribution of all hydrophilic and lipophilic regions over any given shape. The electrostatic potential on the molecular surface is calculated based on the atomic point charges. The resulting hydrophobic moment vector is specific for the instantaneous conformation, and it takes into account all structural characteristics of the molecule, e.g., partial unfolding, bending, and side-chain torsion angles. Extended all-atom molecular dynamics simulations are then used to calculate the equilibrium hydrophobic moments for two antimicrobial peptides, gramicidin S and PGLa, under different conditions. We show that their effective hydrophobic moment vectors reflect the distribution of polar and nonpolar patches on the molecular surface and the calculated electrostatic surface potential. A comparison of simulations in solution and in lipid membranes shows how the peptides undergo internal conformational rearrangement upon binding to the bilayer surface. A good correlation with solid-state NMR data indicates that the hydrophobic moment vector can be used to predict the membrane binding geometry of peptides. This method is available as a web application on http://www.ibg.kit.edu/HM/., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Single D-amino acid substitutions can be used to suppress or slow down the aggregation of peptides into β-sheeted assemblies compared to the respective L-amino acids. Here, we investigate the influence of local stereochemistry in the model peptide [KIGAKI]3-NH2, which is known to form amyloid-like fibrils. To find out whether aggregation plays a role in various biologically relevant functions that involve peptide-lipid interactions, we studied the antimicrobial, hemolytic and fusogenic activities of this amphiphilic membrane-active molecule. The stiff and sterically constrained amino acid CF3-Bpg [3-(trifluoromethyl)-bicyclopent-[1,1,1]-1-ylglycine] was incorporated either as an L- or a D-enantiomer at different hydrophobic positions of the KIGAKI sequence. D-Epimers have a higher aggregation threshold than the L-epimers, yet the aggregation of both was confirmed using electron microscopy and circular dichroism. Solid-state (19)F-NMR analysis showed that the peptide aggregated in native membranes from human erythrocytes and bacterial protoplasts in the same way as in synthetic lipid bilayers. We then monitored the effect of the single L- or D-CF3-Bpg substitutions in KIGAKI on its distinct biological activities, which have to be measured at low peptide concentrations where the aggregation threshold cannot be directly assessed. These functional assays showed that the aggregation propensity of KIGAKI does not play a role in its antimicrobial action, but an increased tendency to aggregate promotes other undesirable effects such as hemolysis and membrane fusion. These results confirm the membranolytic and thereby toxic nature of amyloidogenic peptides, and emphasize the unpredictable role of peptide aggregation in the different assays used to study biological activities.

The membrane alignment of the amphiphilic alpha-helical model peptide MSI-103 (sequence [KIAGKIA]3-NH2) was examined by solid state 2H-NMR in different lipid systems by systematically varying the acyl chain length and degree of saturation, the lipid head group type, and the peptide-to-lipid molar ratio. In liquid crystalline phosphatidylcholine (PC) lipids with saturated chains, the amphiphilic helix changes its orientation from a surface-bound "S-state" to a tilted "T-state" with increasing peptide concentration. In PC lipids with unsaturated chains, on the other hand, the S-state is found throughout all concentrations. Using phosphatidylethanolamine lipids with a small head group or by addition of lyso-lipids with only one acyl chain, the spontaneous curvature of the bilayer was purposefully changed. In the first case with a negative curvature only the S-state was found, whereas in systems with a positive curvature the peptide preferred the obliquely immersed T-state at high concentration. The orientation of MSI-103 thus correlates very well with the shape of the lipid molecules constituting the membrane. Lipid charge, on the other hand, was found to affect only the initial electrostatic attraction to the membrane surface but not the alignment preferences. In bilayers that are "sealed" with 20% cholesterol, MSI-103 cannot bind in a well-oriented manner and forms immobilized aggregates instead. We conclude that the curvature properties of a membrane are a key factor in the interactions of amphiphilic helical peptides in general, whose re-alignment and immersion preferences may thus be inferred in a straightforward manner from the lipid-shape concept.

The functional state of a membrane-active peptide is often defined by its conformation, molecular orientation, and its oligomeric state in the lipid bilayer. These "static" structural properties can be routinely studied by solid state NMR using isotope-labeled peptides. In the highly dynamic environment of a liquid crystalline biomembrane, however, the whole-body fluctuations of a peptide are also of paramount importance, although difficult to address and most often ignored. Yet it turns out that disregarding such motional averaging in calculating the molecular alignment from orientational NMR-constraints may give a misleading, if not false picture of the system. Here, we demonstrate that the reliability of a simplified static or an advanced dynamic data analysis depends critically on the choice of isotope labeling scheme used. Two distinctly different scenarios have to be considered. When the labels are placed on the side chains of a helical peptide (such as a CD(3)- or CF(3)-group attached to the C(alpha)C(beta) bond), their nuclear spin interaction tensors are very sensitive to motional averaging. If this effect is not properly accounted for, the helix tilt angle tends to be severely underestimated. At the same time, the analysis of labels in the side chains allows to extract valuable dynamical information about whole-body fluctuations of the peptide helix in the membrane. On the other hand, the alternative labeling scheme where (15)N-labels are accommodated within the peptide backbone, will yield nearly correct helix tilt angles, irrespective as to whether dynamics are taken into account or not., (Copyright 2009 Elsevier B.V. All rights reserved.)

Membrane proteins and peptides exhibit a preferred orientation in the lipid bilayer while fluctuating in an anisotropic manner. Both the orientation and the dynamics have direct functional implications, but motions are usually not accessible, and structural descriptions are generally static. Using simulated data, we analyze systematically the impact of whole-body motions on the peptide orientations calculated from two-dimensional polarization inversion spin exchange at the magic angle (PISEMA) NMR. Fluctuations are found to have a significant effect on the observed spectra. Nevertheless, wheel-like patterns are still preserved, and it is possible to determine the average peptide tilt and azimuthal rotation angles using simple static models for the spectral fitting. For helical peptides undergoing large-amplitude fluctuations, as in the case of transmembrane monomers, improved fits can be achieved using an explicit dynamics model that includes Gaussian distributions of the orientational parameters. This method allows extracting the amplitudes of fluctuations of the tilt and azimuthal rotation angles. The analysis is further demonstrated by generating first a virtual PISEMA spectrum from a molecular dynamics trajectory of the model peptide, WLP23, in a lipid membrane. That way, the dynamics of the system from which the input spectrum originates is completely known at atomic detail and can thus be directly compared with the dynamic output obtained from the fit. We find that fitting our dynamics model to the polar index slant angles wheel gives an accurate description of the amplitude of underlying motions, together with the average peptide orientation.

Solid-state (2)H-NMR is routinely used to determine the alignment of membrane-bound peptides. Here we demonstrate that it can also provide a quantitative measure of the fluctuations around the distinct molecular axes. Using several dynamic models with increasing complexity, we reanalyzed published (2)H-NMR data on two representative alpha-helical peptides: 1), the amphiphilic antimicrobial peptide PGLa, which permeabilizes membranes by going from a monomeric surface-bound to a dimeric tilted state and finally inserting as an oligomeric pore; and 2), the hydrophobic WALP23, which is a typical transmembrane segment, although previous analysis had yielded helix tilt angles much smaller than expected from hydrophobic mismatch and molecular dynamics simulations. Their (2)H-NMR data were deconvoluted in terms of the two main helix orientation angles (representing the time-averaged peptide tilt and azimuthal rotation), as well as the amplitudes of fluctuation about the corresponding molecular axes (providing the dynamic picture). The mobility of PGLa is found to be moderate and to correlate well with the respective oligomeric states. WALP23 fluctuates more vigorously, now in better agreement with the molecular dynamics simulations and mismatch predictions. The analysis demonstrates that when (2)H-NMR data are fitted to extract peptide orientation angles, an explicit representation of the peptide rigid-body angular fluctuations should be included.

The antimicrobial activity of amphipathic alpha-helical peptides is usually attributed to the formation of pores in bacterial membranes, but direct structural information about such a membrane-bound state is sparse. Solid state (2)H-NMR has previously shown that the antimicrobial peptide PGLa undergoes a concentration-dependent realignment from a surface-bound S-state to a tilted T-state. The corresponding change in helix tilt angle from 98 to 125 degrees was interpreted as the formation of PGLa/magainin heterodimers residing on the bilayer surface. Under no conditions so far, has an upright membrane-inserted I-state been observed in which a transmembrane helix alignment would be expected. Here, we have demonstrated that PGLa is able to assume such an I-state in a 1:1 mixture with magainin 2 at a peptide-to-lipid ratio as low as 1:100 in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol model membranes. This (2)H-NMR analysis is based on seven orientational constraints from Ala-3,3,3-d(3) substituted in a non-perturbing manner for four native Ala residues as well as two Ile and one Gly. The observed helix tilt of 158 degrees is rationalized by the formation of heterodimers. This structurally synergistic effect between the two related peptides from the skin of Xenopus laevis correlates very well with their known functional synergistic mode of action. To our knowledge, this example of PGLa is the first case where an alpha-helical antimicrobial peptide is directly shown to assume a transmembrane state that is compatible with the postulated toroidal wormhole pore structure.

The cationic antimicrobial peptide PGLa is electrostatically attracted to bacterial membranes, binds as an amphiphilic alpha-helix, and is thus able to permeabilize the lipid bilayer. Using solid state (2)H-NMR of non-perturbing Ala-d(3) labels on the peptide, we have characterized the helix alignment under a range of different conditions. Even at a very high peptide-to-lipid ratio (1:20) and in the presence of negatively charged lipids, there was no indication of a toroidal wormhole structure. Instead, PGLa re-aligns from a surface-bound S-state to an obliquely tilted T-state, which is presumably dimeric. An intermediate structure half-way between the S- and T-state was observed in fully hydrated multilamellar DMPC vesicles at 1:50, suggesting a fast exchange between the two states on the time scale of >50 kHz. We demonstrate that this equilibrium is shifted from the S- towards the T-state either upon (i) increasing the peptide concentration, (ii) adding negatively charged DMPG, or (iii) decreasing the level of hydration. The threshold concentration for re-alignment in DMPC is found to be between 1:200 and 1:100 in oriented samples at 96% humidity. In fully hydrated multilamellar DMPC vesicles, it shifts to an effective peptide-to-lipid ratio of 1:50 as some peptides are able to escape into the bulk water phase.

Solid-state NMR methods employing (2)H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane alpha-helical peptide acetyl-GWW(LA)(8)LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane alpha-helical conformation. A small tilt angle of 4.5 degrees was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2 degrees in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA)(6)LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method.

Ternary phase diagrams have been constructed of systems with dioleoylphosphatidylcholine (DOPC) and water, and two alpha-helical membrane-spanning model peptides, KKLAKK16[KK(LA)6KK] and KKLAKK20[KK(LA)8KK]. It was found that these peptides induced non-lamellar liquid crystalline phases. The amount of peptide needed for this phase transition depended on the water content and the temperature; and for KKLAKK16, a smaller amount of peptide was needed to induce non-lamellar phases than for KKLAKK20. Both peptides were found to induce an isotropic phase, and KKLAKK16 also induced a reversed hexagonal phase. Both peptides may also reside in a lamellar (L(alpha)) phase. When magic angle spinning (MAS) 31P NMR experiments were performed on samples containing the L(alpha) phase and an isotropic phase, four different isotropic chemical shifts were observed. The isotropic chemical shifts could be assigned to the phases, using spinning sidebands to calculate the chemical shift anisotropy (CSA) corresponding to each isotropic shift. MAS 13C NMR also indicated a difference in the aggregational state of the peptides between the L(alpha) and isotropic phases. The phase diagrams were compared to the phase diagram of a similar model peptide, AWW(LA)5WWA in systems with DOPC and water. It was concluded that the phase behaviour was influenced by both electrostatic interactions between the peptides and the lipid headgroups, and the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer.

The effect of solubilized hydrophobic peptides on the phase behavior of dioleoylphosphatidylcholine (DOPC)/water system was studied by 2H- and 31P-NMR spectroscopy and by x-ray diffraction, and partial phase diagrams were constructed. The utilized peptides were HCO-AWW(LA)5WWA-NHCH2CH2OH (WALP16), which is an artificial peptide designed to resemble a transmembrane part of a membrane protein; and VEYAGIALFFVAAVLTLWSMLQYLSAAR (Pgs peptide E), a peptide that is identical to one of the putative transmembrane segments of the membrane-associated protein phosphatidylglycerophosphate synthase (Pgs) in Escherichia coli. Circular dichroism spectroscopy suggests that both peptides are mostly alpha-helical in DOPC vesicles. The most striking features in the phase diagram of the WALP16/DOPC/water system are 1) a single lamellar liquid crystalline (L alpha) phase forms only at very low peptide concentrations. 2) At low water content and above a peptide/lipid molar ratio of approximately 1:75 a reversed hexagonal liquid crystalline (H[II]) phase coexists with an L alpha phase, while in excess water this phase forms at a peptide/lipid molar ratio of approximately 1:25. 3) At peptide/lipid ratios > or =1:6 a single H(II) phase is stable. Also, the Pgs peptide E strongly affects the phase behavior, and a single L alpha phase is only found at low peptide concentrations (peptide/lipid molar ratios <1:50), and water concentrations <45% (w/w). Higher peptide content results in coexistence of L alpha and isotropic phases. Generally, the fraction of the isotropic phase increases with increasing temperature and water concentration, and at 80% (w/w) water content only a single isotropic phase is stable at 55 degrees C. Thus, both peptides were found to be able to induce nonlamellar phases, although different in structure, in the DOPC/water system. The phase transitions, the extensions of the one-phase regions, and the phase structures observed for the two systems are discussed in terms of the molecular structure of the two peptides and the matching between the hydrophobic lengths of the peptides and the bilayer thickness of DOPC.