1H-NMR of reverse micelles. I: The surfactant resonances as probes for the AOT/water/isooctane system

1H-NMR spectra of Aerosol-OT (AOT) reverse micelles in isooctane are reported at various water contents and several temperatures. The resonances from the AOT protons near the polar head are completely assigned. The NMR parameters (chemical shift and linewidth) appear to be suitable probes for characterizing the physico-chemical state of micelles, in particular in order to define the range in which the system is fully homogeneous and transparent. The results correlate well with those obtained from optical density measurements. Lanthanide ions dissolved in the water phase selectively perturb the AOT resonances from the protons nearest to the polar head; conversely, non-polar shift reagents soluble in the organic phase do not cause appreciable effects on any of the observable signals.     

Conformations of model peptides in membrane-mimetic environments.

The influence of a membrane environment on the conformational energetics of a polypeptide chain has been investigated through studies of model peptides in a variety of membrane-mimetic media. Nuclear magnetic resonance (NMR) and circular dichroism (CD) data have been obtained for the peptides in bulk hydrophobic solvents, normal micelles, and reversed micelles. Several hydrophobic peptides which are sparingly soluble in water have been solubilized in aqueous sodium dodecyl sulfate (SDS) solution. NMR and CD data indicate that the micelle-solubilized peptides experience an environment with the conformational impact of bulk methanol, and have decreased conformational freedom. The site of residence of the peptides interacting with the micelles appears to be near the surfactant head groups, in a region permeated by water, and not in the micelle core. Strongly hydrophilic peptides have been solubilized in nonpolar solvents by reversed micelles. These peptides are located in small water pools in close association with the head groups of the surfactant. NMR and CD data show that there is a conformational impact of this interfacial water region on peptide solubilizates distinct from that of bulk water.     

Localization and interaction of genistein with model membranes formed with dipalmitoylphosphatidylcholine (DPPC)

The effect of genistein on the liposomes formed with dipalmitoylphosphatidylcholine was studied with the application of Fourier-transform infrared spectroscopy, nuclear magnetic resonance ((1)H NMR) and electron paramagnetic resonance techniques. Membranous structures organization of human skin fibroblasts and colon myofibroblasts was also examined using fluorescence and electron microscopy. The strongest rigidifying effect of genistein with respect to polar head groups was concluded on the basis of the effect of the flavonoid on the shape of NMR lines attributed to -N(+)(CH(3))(3) groups. The rigidifying effect of genistein with respect to the hydrophobic core of lipid membranes was also concluded from the genistein-dependent broadening of the NMR lines assigned to -CH(2) groups and terminal -CH(3) groups of alkyl chains. EPR data supported ordering effect of genistein of the hydrophobic core in the liquid-crystalline phase (L(α)). The analysis of the FTIR spectra of the two-component liposomes showed that genistein incorporates into DPPC membranes via hydrogen bonding between the lipid polar head groups in the C-O-P-O-C segment and its hydroxyl groups. Both fluorescence microscopy and ultrastructural observation revealed changes in membranous structures organization as aftermath of genistein treatment. In conclusion, genistein localized within membranes changes the properties of membrane that can be followed by the changes inside cells being crucial for pharmacological activity of genistein used in cancer or other disease treatment.     

Interaction of piroxicam with micelles: effect of hydrophobic chain length on structural switchover

Molecules, whose pK(a) values can be easily fine-tuned by their microenvironment, are expected to be profoundly affected by the heterogeneous environments of membranes. Membrane parameters can have a strong effect in choosing a particular structural form of a molecule for incorporation/interaction. A case study has been presented for piroxicam, a non-steroidal anti-inflammatory drug of oxicam group, whose targets are cyclooxygenases, which are membrane active proteins. The structural dynamism of piroxicam is reflected in the ease with which it can switchover or convert from one prototropic form to the other guided by its environment. In this work we have studied the effect of varying hydrophobic chain length and surface charges in fine-tuning the interaction of piroxicam with micelles. Interaction of piroxicam with three types of micelles with identical negatively charged head groups and varying tail lengths viz., sodium dodecyl sulfate (S12S), sodium decyl sulfate (S10S) and sodium octyl sulfate (S8S) shows that there is a shift in the apparent pK(a) in the direction that favors the switchover or conversion from the anionic form to the global neutral form. The binding constants of piroxicam with three micelles show a linear dependence on chain length. Interaction was also studied with micelles having oppositely charged head groups and different chain lengths viz., dodecyl N,N,N-trimethyl ammonium bromide (DTAB) and cetyl N,N,N-trimethyl ammonium bromide (CTAB). For micelles having identical chain lengths but oppositely charged head groups viz., S12S and DTAB, pK(a) shifts in two opposite directions compared to that in the absence of any surfactant. This is expected when electrostatic force is the only driving force. This case study demonstrates the effect of hydrophobic chain length and surface charges in fine-tuning the equilibrium between different structural forms of piroxicam. Our results also imply that for structurally dynamic drugs like piroxicam the nature of the biomembranes, characterized by different membrane parameters, should play a crucial role in choosing a particular structural form of the drug that will be finally presented to their targets.     

Unsaturation at the surfactant head: influence on the activity of lipase and horseradish peroxidase in reverse micelles

Influence of unsaturation present at the surfactant head on the activity of interfacially located enzyme, lipase, and horseradish peroxidase (HRP) is investigated in cationic reverse micelles of a series of surfactants having unsaturated (allyl and pyridinium moieties) as well as analogous saturated (n-propyl and piperidinium moieties) polar head. Lipase activity increases with n-propyl (saturated) substitution as the increase in the headgroup area (A(min)) presumably provides greater space for the enzyme to attain flexible conformation and increases the local concentrations of enzyme and substrate at the interface. In contrast, activity of lipase decreases with increasing number of allyl (unsaturated) substitution though A(min) gradually increased. Similar trend in deactivation was observed when unsaturation is present in cyclic ring (pyridine) at the surfactant head in comparison to the saturated analogue, piperidine. Circular dichroism (CD) spectra of lipase in reverse micelles indicate that ellipticity in the far-UV region increases with increasing unsaturation. Thus, lipase probably loses its alpha-helix content and thereby its activity. Inhibition of biocatalyst with increasing unsaturation at the polar head of surfactant is also observed in case of HRP, an oxidoreductase enzyme.     

Characterization of the interaction between surfactants and enzymes by fluorescence probe

In order to investigate the mechanism of the different stimulatory effects of the biosurfactant rhamnolipid and the chemical surfactant Tween 80 on enzymatic hydrolysis of lignocellulose, the interaction between surfactants and enzymes was analyzed by the fluorescence probe method using pyrene as probe. Based on the evolution law of pyrene fluorescence spectroscopy in the “surfactants-enzymes” systems, the interaction relationship between surfactants and enzymes was analyzed and discussed in this paper. The results show that enzyme molecules bind with rhamnolipid molecules, participate in the formation of rhamnolipid micelles, and increase the inner hydrophobic polarity of micelles, but do not change the properties of rhamnolipid micelles above the CMC (Critical Micelle Concentration). Nevertheless, for Tween 80, enzyme molecules also participate in the forming of micelles, however, they exhibit a stronger interaction with enzymes above the CMC. Both rhamnolipid and Tween 80 bind more strongly with xylanase than cellulase. Considering also previous experimental results, it can be concluded that the interaction between surfactants and enzymes improve enzyme stability and activity, and, therefore, the efficiency of enzymatic hydrolysis of lignocellulose is enhanced. The findings further provide theoretical knowledge about the mechanism of the stimulative effects of surfactants on enzymatic hydrolysis of lignocellulose.     

Cholate-induced dimerization of detergent- or phospholipid-solubilized bovine cytochrome C oxidase

Bovine heart cytochrome c oxidase (CcO), solubilized by either nonionic detergents or phospholipids, completely dimerizes upon the addition of bile salts, e.g., sodium cholate, sodium deoxycholate, or CHAPS. Bile salt induced dimerization occurs whether dodecyl maltoside, decyl maltoside, or Triton X-100 is the primary solubilizing detergent or the enzyme is dispersed in phosphatidylcholine, phosphatidylethanolamine, or mixtures thereof. In each case, complete CcO dimerization can be verified by sedimentation velocity and sedimentation equilibrium after correction for bound detergent and/or phospholipid. The relative concentration of the bile salt is critical for production of homogeneous, dimeric CcO. For example, enzyme solubilized by 2 mM detergent requires an equal molar concentration of sodium cholate. Similarly, enzyme dispersed in 20 mM phospholipid requires 50 mM sodium cholate, concentrations that are commonly used to reconstitute CcO into small unilamellar vesicles. Bile salts do more than just stabilize dimeric CcO and prevent detergent-induced dissociation into monomers. They are able to completely reverse detergent-induced monomerization and cause completely monomeric CcO to reassociate. Dimeric CcO so generated is no more stable than the original complex and easily dissociates into monomers if the bile salt is removed. The dimerization process is dependent upon a full complement of subunits; e.g., if subunits VIa and VIb are removed, the resulting monomeric CcO will not reassociate upon the addition of sodium cholate. These results support four important consequences: (1) dissociation of dimeric CcO into monomers is reversible; (2) stable dimers can be produced under solution conditions; (3) dimers can be stabilized even at relatively high pH and low enzyme concentration; and (4) subunits VIa and VIb are required for dimerization.     

Reactivity of trypsin in reverse micelles: neglected role of aggregate size compared to water-pool components

The catalytic efficiency of trypsin was estimated in cationic reverse micelles as a function of the concentration of water-pool components and aggregate size to determine their independent influence on enzyme activity. The variation in the aggregate size/water-pool size was achieved by changing both the W0 (mole ratio of water to surfactant) and the headgroup area of surfactant through introduction of hydroxyethyl groups at the polar head. The local molar concentrations of water present inside the water-pool ([H2O]wp) of different cationic reverse micelles across varying W0 was estimated using a modified phenyl cation-trapping protocol. The [H2O]wp in cationic reverse micelles (surfactant/isooctane/n-hexanol/water) increases with W0 and attains the molarity of normal water beyond W0=40 irrespective of the nature of headgroup. Concurrently, the catalytic activity of trypsin compartmentalized within the water-pool increases with the increase in [H2O]wp upto an optimal W0=40 in organized solutions of any surfactant. The aggregate size (determined by static light scattering) also increases expectedly with W0 and noticeably with the area of the surfactant headgroup at similar W0. Since the enzyme activity rises both with the increase in water-pool size and [H2O]wp, trypsin's efficiency was compared with these two parameters across reverse micelles of varying surfactant headgroup size at similar W0 to determine their probable independent influence in regulating the enzyme activity. Noticeably, the efficiency of trypsin rises two to ninefold in spite of the [H2O]wp being distinctly lower in case of hydroxyethyl group substituted surfactants compared to cetyltrimethylammonium bromide w/o microemulsions at similar W0. Thus, the influence of the aggregate size possibly plays an important role alongwith the [H2O]wp in modulating the enzyme activity.     

Binding of alkyl polyglucoside surfactants to bacteriorhodopsin and its relation to protein stability

The binding of alkyl polyglucoside surfactants to the integral membrane protein bacteriorhodopsin (BR) and the formation of protein-surfactant complexes are investigated by sedimentation equilibrium via analytical ultracentrifugation and by small-angle neutron scattering (SANS). Contrast variation techniques in SANS enable measurement of the composition of the protein-surfactant complexes and determination of the thickness of the surfactant shell bound to the protein. The results indicate that alkyl polyglucosides can bind to BR as single surfactant layers or as a thicker shell. The thickness of the surfactant shell increases with increasing surfactant tail length, and it is generally unrelated to the aggregation number of the micelles even for a small and predominantly hydrophobic membrane protein such as BR. The aggregation numbers determined by sedimentation equilibrium methods match those measured by SANS, which also allows reconstruction of the shape of the protein-detergent complex. When the surfactant is present as a single layer, the BR loses activity, as measured by absorption spectroscopy, more quickly than it does when the surfactant forms a thicker shell.     

Delta-opiate DPDPE in magnetically oriented phospholipid micelles: binding and arrangement of aromatic pharmacophores

D-Penicillamine(2,5)-enkephalin (DPDPE) is a potent opioid peptide that exhibits a high selectivity for the delta-opiate receptors. This zwitterionic peptide has been shown, by pulsed-field gradient 1H NMR diffusion studies, to have significant affinity for a zwitterionic phospholipid bilayer. The bilayer lipid is in the form of micelles composed of dihexanoylphosphatidylcholine (DHPC) and dimyristoylphosphatidylcholine (DMPC) mixtures, where the DMPC forms the bilayer structure. At high lipid concentration (25% w/w) these micelles orient in the magnetic field of an NMR spectrometer. The resulting 1H-13C dipolar couplings and chemical shift changes in the natural abundance 13C resonances for the Tyr and Phe aromatic rings were used to characterize the orientations in the bilayer micelles of these two key pharmacophores.     

Two sites of interaction of anions with cytochrome a in oxidized bovine cytochrome c oxidase

An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.     

An NMR study of pyridine associated with DMPC liposomes and magnetically ordered DMPC-surfactant mixed micelles.

With molecular dynamics simulations of phospholipid membranes becoming a reality, there is a growing need for experiments that provide the molecular details necessary to test these computational results. Pyridine is used here to explore the interaction of planar aromatic groups with the water-lipid interface of membranes. It is shown by magic angle spinning 13C nuclear magnetic resonance (NMR) to bind between the glycerol and choline groups of dimyristoylphosphatidylcholine (DMPC) liposomes. The axial pattern for the 31P NMR spectrum of DMPC liposomes is preserved even with more than half of the interfacial sites occupied, indicating that pyridine does not disrupt the lamellar phase of this lipid. 2H NMR experiments of liposomes in deuterium oxide demonstrate that pyridine might promote greater penetration of water into restricted regions in the interface. Magnetically oriented DMPC/surfactant micelles were investigated as a means for improving resolution and sensitivity in NMR studies of species bound to bilayers. The quadrupolar splittings in the 2H NMR spectra of d5-pyridine in DMPC liposomes and magnetically oriented DMPC/Trixon X-100 micelles indicate a common bound state for the two bilayer systems. The well resolved quadrupolar splittings of d5-pyridine in oriented micelles were used to establish the tilt of the pyridine ring relative to the bilayer plane.     

Escherichia coli diacylglycerol kinase: a case study in the application of solution NMR methods to an integral membrane protein

Diacylglycerol kinase (DAGK) is a 13-kDa integral membrane protein that spans the lipid bilayer three times and which is active in some micellar systems. In this work DAGK was purified using metal ion chelate chromatography, and its structural properties in micelles and organic solvent mixtures studies were examined, primarily to address the question of whether the structure of DAGK can be determined using solution NMR methods. Cross-linking studies established that DAGK is homotrimeric in decyl maltoside (DM) micelles and mixed micelles. The aggregate detergent-protein molecular mass of DAGK in both octyl glucoside and DM micelles was determined to be in the range of 100-110 kDa-much larger than the sum of the molecular weights of the DAGK trimers and the protein-free micelles. In acidic organic solvent mixtures, DAGK-DM complexes were highly soluble and yielded relatively well-resolved NMR spectra. NMR and circular dichroism studies indicated that in these mixtures the enzyme adopts a kinetically trapped monomeric structure in which it irreversibly binds several detergent molecules and is primarily alpha-helical, but in which its tertiary structure is largely disordered. Although these results provide new information regarding the native oligomeric state of DAGK and the structural properties of complex membrane proteins in micelles and organic solvent mixtures, the results discourage the notion that the structure of DAGK can be readily determined at high resolution with solution NMR methods.     

Ethylene glycol and the thermostability of trypsin in a reverse micelle system

The influence of ethylene glycol (EG) on the kinetics of hydrolysis of N-alpha-benzoyl-L-arginine ethyl ether catalyzed by trypsin encapsulated in sodium bis-(2-ethylhexyl)sulfosuccinate (AOT)-based reverse micelles was studied at different temperatures. Ethylene glycol was shown to shift the range of the trypsin activity in the reverse micelles towards higher temperatures. Infrared spectroscopy showed a stabilizing effect of EG on the secondary structure of the protein in the system of reverse micelles. Electron spin resonance spectroscopy showed that the solubilized protein affected the interactions of EG with the polar head groups of AOT and altered the rigidity of the micellar matrix. The results indicate that EG increases the thermostability of the solubilized enzyme in microemulsion media by two mechanisms.     

FTIR, 1H NMR and EPR spectroscopy studies on the interaction of flavone apigenin with dipalmitoylphosphatidylcholine liposomes

Apigenin (5,7,4′-trihydroxyflavone) is a cancer chemopreventive agent and a member of the family of plant flavonoids. Apigenin interaction with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was investigated by means of FTIR spectroscopy, ¹H NMR and EPR techniques. Fluorescent microscopy and electron microscopy were applied to study the apigenin effects on colon myofibroblasts and human skin fibroblasts. The strong rigidifying effect of apigenin with respect to polar head groups was concluded on the basis of the action of the flavone on partition coefficient of Tempo spin label between the water and lipid phases. The ordering effect was also found in hydrophobic region at the depth monitored by 5-SASL and 16-SASL spin labels. The inclusion of apigenin to the membrane restricted the motional freedom of polar head groups lowering penetration of Pr^3 + ions to the membranes. The ¹H NMR technique supported also the restriction of motional freedom of the membrane in the hydrophobic region, especially in the zone of CH₂ groups of alkyl chains. FTIR analysis showed that apigenin incorporates into DPPC liposomes via hydrogen bonding between its own hydroxyl groups and lipid polar head groups in the COPOC segment. It is also very likely that hydroxyl groups of apigenin link with polar groups of DPPC by water bridges. Electron and fluorescence microscopic observations revealed changes in the internal membrane organization of the examined cells. In conclusion, the changes of the structural and dynamic properties of membranes can be crucial for processes involving tumor suppression signal transduction pathways and cell cycle regulation.     

Interaction of myelin basic protein with micelles of dodecylphosphocholine

Interactions of myelin basic protein (MBP) and peptides derived from it with micelles of dodecylphosphocholine (DPC) and perdeuterated DPC have been studied by proton nuclear magnetic resonance (NMR) at 400 MHz and by circular dichroism (CD). When MBP binds to DPC micelles, it acquires about 18% alpha-helicity. The CD spectra of various peptides derived by cleavage of MBP indicate that a major alpha-helical region occurs in residues 85-99 just before the sequence of three prolyl residues 100-102. From line broadenings by fatty acid spin-labels in the micelles and from changes in chemical shifts, the NMR data identify specific residues in MBP that participate in lipid binding. One such sequence is an alpha-helical region from residues 85 to 95, and others occur around methionine-21 and between residues 117 and 135. The different effects of C5, C12, and C16 spin-labels suggest that some segments of the protein may penetrate beyond the dipolar interfacial region of the micelles into the hydrophobic interior, but no part of the protein is protected by the micelles against rapid exchange of its amide groups with the aqueous environment. Even at a lipid to protein molar ratio of 200/1, most NMR resonances from side chains of amino acid residues are not appreciably broadened, suggesting that much of the polypeptide remains highly mobile.     

Dependence of the conformation of the polar head groups of phosphatidylcholine on its packing in bilayers. Nuclear magnetic resonance studies on the effect of the binding of lanthanide ions.

Proton magnetic resonance spectra of vesicles of various sizes composed of egg phosphatidylcholine (PC) with varying concentrations of cholesterol differed in the apparent line width of the signal of the methylene protons of PC (delta v1/2). They also varied in the extent of lanthanide-induced shifts of the 31P and 1H NMR signals of the corresponding nuclei of the polar head groups located on the outer surface of the vesicles (delta delta). The differences in the lanthanide-induced shifts of the 31P signals are fully accounted for by the ratio between the externally added lanthanide and the number of PC head groups available for interaction with the lanthanide ions. This was not the case ofr the changes in the 1H NMR spectra. Here delta delta decreased with increasing delta v1/2, suggesting that the packing of the PC paraffinic chaings in the bilayer affects the conformation of the polar head groups; tightening of the packing probably results in a more extended conformation of the head groups. This conclusion is also supported by the larger effect lanthanides have on the 1H chemical shift of the choline head groups on the outer surface of small unilamellar vesicles as compared to groups on the inner, tighter packed layer.     

Characterisation of the interaction of the C-terminus of the dopamine D2 receptor with neuronal calcium sensor-1

NCS-1 is a member of the neuronal calcium sensor (NCS) family of EF-hand Ca(2+) binding proteins which has been implicated in several physiological functions including regulation of neurotransmitter release, membrane traffic, voltage gated Ca(2+) channels, neuronal development, synaptic plasticity, and learning. NCS-1 binds to the dopamine D2 receptor, potentially affecting its internalisation and controlling dopamine D2 receptor surface expression. The D2 receptor binds NCS-1 via a short 16-residue cytoplasmic C-terminal tail. We have used NMR and fluorescence spectroscopy to characterise the interactions between the NCS-1/Ca(2+) and D2 peptide. The data show that NCS-1 binds D2 peptide with a K(d) of ～14.3 μM and stoichiometry of peptide binding to NCS-1 of 2:1. NMR chemical shift mapping confirms that D2 peptide binds to the large, solvent-exposed hydrophobic groove, on one face of the NCS-1 molecule, with residues affected by the presence of the peptide spanning both the N and C-terminal portions of the protein. The NMR and mutagenesis data further show that movement of the C-terminal helix 11 of NCS-1 to fully expose the hydrophobic groove is important for D2 peptide binding. Molecular docking using restraints derived from the NMR chemical shift data, together with the experimentally-derived stoichiometry, produced a model of the complex between NCS-1 and the dopamine receptor, in which two molecules of the receptor are able to simultaneously bind to the NCS-1 monomer.     

Lipid requirements of the plasma membrane ATPases from oat roots and yeast

The lipid specificity of the plasma membrane ATPases from oat roots and yeast has been investigated by reconstituting delipidated enzyme with phospholipid vesicles and with micelles of lysophospholipids and other detergents. The plant ATPase is activated by Triton X-100 and by all phospholipid and lysophospholipid species, exhibiting only a slight preference for zwitterionic polar heads (phosphorylcholine and phosphorylethanolamine). No unsaturation is required on the hydrophobic chain. On the other hand, the yeast ATPase requires a negatively charged polar head (with preference for phosphorylglycerol and phosphorylinositol) and an unsaturated hydrophobic chain.     

Structures and mode of membrane interaction of a short alpha helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy.

The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as alpha helices or beta sheets. We have shown in previous studies that incorporating approximately 30%d-amino acids into a short alpha helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating d-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic alpha helical cytolytic peptide KLLLKWLL KLLK-NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the alpha helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of alpha helical or beta sheet structures.