Binding mode of a cationic porphyrin to parallel and antiparallel thrombin binding aptamer G-quadruplex

Abstract

The spectral properties of meso-tetrakis (N-methylpyridinium-4-yl)porphyrin (TMPyP) in the presence of parallel and antiparallel G-quadruplexes formed from a thrombin-binding aptamer G-quadruplex (5′-G₃T₂G₃TGTG₃T₂G₃) were investigated in this study. Red shift and hypochromism in the Soret absorption band of TMPyP were observed after binding to both parallel and antiparallel G-quadruplexes. The extent of changes in the absorption spectra were similar for both conformers. No circular dichroism spectrum was induced in the Soret region for both parallel and antiparallel G-quadruplexes. This is suggest that there is no or very weak interaction between electric transitions of nucleobases and porphyrin molecule. The accessibility of the neutral quencher I₂ to the G-quadruplex-bound TMPyP was similar for both parallel and antiparallel G-quadruplexes. All these observations suggest that TMPyP was bound at the outside of the quadruplexes, and conceivably interacted with the phosphate group via a weak electrostatic interaction.

Communicated by Ramaswamy H. Sarma     

Visual observation of G-quadruplex DNA with the label-free fluorescent probe silole with aggregation-induced emission

AIE molecule silole 1 could be used to detect G-quadruplex formation using an exonuclease I hydrolysis assay. This visual observation of G-quadruplexes has been successfully used in investigating multiple G-quadruplexes, including the one-stranded telomeric, c-myc, c-kit, VEGF G-quadruplexes, and a d(G₄T₄G₄) inter-molecular G-quadruplex. The detection of G-quadruplex can be also applied to G-quadruplex isomers induced by small molecules.     

Structural transition from antiparallel to parallel G-quadruplex of d(G4T4G4) induced by Ca2+

Guanine quadruplex (G-quadruplex) structures are formed by guanine-rich oligonucleotides. Because of their in vivo and in vitro importance, numerous studies have been demonstrated that the structure and stability of the G-quadruplex are dependent on the sequence of oligonucleotide and environmental conditions such as existing cations. Previously, we quantitatively investigated the divalent cation effects on the antiparallel G-quadruplex of d(G₄T₄G₄), and found that Ca²⁺ induces a structural transition from the antiparallel to parallel G-quadruplex, and finally G-wire formation. In the present study, we report in detail the kinetic and thermodynamic analyses of the structural transition induced by Ca²⁺ using stopped-flow apparatus, circular dichroism, size-exclusion chromatography (SEC) and atomic force microscopy. The quantitative parameters showed that at least two Ca²⁺ ions were required for the transition. The kinetic parameters also indicated that d(G₄T₄G₄) underwent the transition through multiple steps involving the Ca²⁺ binding, isomerization and oligomerization of d(G₄T₄G₄). The parallel-stranded G-wire structure of d(G₄T₄G₄), which is a well controlled alignment of numerous DNA strands with G-quartets, as the final product induced by Ca²⁺, was observed using SEC and atomic force microscopy. These results provide insight into the mechanism of the structural transition and G-wire formation and are useful for constructing a nanomaterial regulated by Ca²⁺.     

Solution structure of a DNA quadruplex containing ALS and FTD related GGGGCC repeat stabilized by 8-bromodeoxyguanosine substitution

A prolonged expansion of GGGGCC repeat within non-coding region of C9orf72 gene has been identified as the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are devastating neurodegenerative disorders. Formation of unusual secondary structures within expanded GGGGCC repeat, including DNA and RNA G-quadruplexes and R-loops was proposed to drive ALS and FTD pathogenesis. Initial NMR investigation on DNA oligonucleotides with four repeat units as the shortest model with the ability to form an unimolecular G-quadruplex indicated their folding into multiple G-quadruplex structures in the presence of K⁺ ions. Single dG to 8Br-dG substitution at position 21 in oligonucleotide d[(G₄C₂)₃G₄] and careful optimization of folding conditions enabled formation of mostly a single G-quadruplex species, which enabled determination of a high-resolution structure with NMR. G-quadruplex structure adopted by d[(G₄C₂)₃GG^BrGG] is composed of four G-quartets, which are connected by three edgewise C-C loops. All four strands adopt antiparallel orientation to one another and have alternating syn-anti progression of glycosidic conformation of guanine residues. One of the cytosines in every loop is stacked upon the G-quartet contributing to a very compact and stable structure.     

Conformational organizations of G-quadruplexes composed of d(G4Tn)3G4

Structural polymorphism is one of the important issues with regard to G-quadruplexes because the structural diversity may significantly affect their biological functions in vivo and their physical property in nano-material. A series of oligonucleotides with four repeat guanines sequence [d(G₄T_n)₃G₄ (n = 1–6)] were designed. In this study, the effects of loop length on the formation of structures of G-quadruplex were investigated through the result of CD (circular dichroism) and 20% non-denatured polyacrylamide gel electrophoresis. Our studies demonstrate that the length of loop in 100 mM KCl solution could predict the conformation of G-quadruplex.     

Induction of G-quadruplex DNA structure by Zn(II) 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin

G-quadruplexes (GQ) are formed by the association of guanine-rich stretches of DNA. Certain small molecules can influence kinetics and thermodynamics of this association. Understanding the mechanism of ligand-assisted GQ folding is necessary for the design of more efficient cancer therapeutics. The oligonucleotide d(TAGGG)₂ forms parallel bimolecular GQ in the presence of ≥66 mM K⁺; GQs are not formed under Na⁺, Li⁺ or low K⁺ conditions. The thermodynamic parameters for GQ folding at 60 μM oligonucleotide and 100 mM KCl are ΔH = −35 ± 2 kcal mol⁻¹ and ΔG₃₁₀ = −1.4 kcal mol⁻¹. Quadruplex [d(TAGGG)₂]₂ binds 2-3 K⁺ ions with K_d of 0.5 ± 0.2 mM. Our work addresses the question of whether metal free 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) and its Zn(II), Cu(II), and Pt(II) derivatives are capable of facilitating GQ folding of d(TAGGG)₂ from single stranded, or binding to preformed GQ, using UV-vis and circular dichroism (CD) spectroscopies. ZnTMPyP4 is unique among other porphyrins in its ability to induce GQ structure of d(TAGGG)₂, which also requires at least a low amount of potassium. ZnTMPyP4 binds with 2:1 stoichiometry possibly in an end-stacking mode with a ∼10⁶ M⁻¹ binding constant, determined through UV-vis and ITC titrations. This process is entropically driven and has ΔG₂₉₈ of −8.0 kcal mol⁻¹. TMPyP4 binds with 3:1 stoichiometry and K_a of ∼10⁶ M⁻¹. ZnTMPyP4 and TMPyP4 are efficient stabilizers of [d(TAGGG)₂]₂ displaying ΔT_1/2 of 13.5 and 13.8 °C, respectively, at 1:2 GQ to porphyrin ratio; CuTMPyP4 shows a much weaker effect (ΔT_1/2 = 4.7 °C) and PtTMPyP4 is weakly destabilizing (ΔT_1/2 = −2.9 °C). The selectivity of ZnTMPyP4 for GQ versus dsDNA is comparable to that of TMPyP4. The ability of ZnTMPyP4 to bind and stabilize GQ, to induce GQ formation, and speed up its folding may suggest an important biological activity for this molecule.     

Effects of deficient of the Hoogsteen base-pairs on the G-quadruplex stabilization and binding mode of a cationic porphyrin

BackgroundIn stabilization of the G-quadruplex, formation of a Hoogsteen base-pair between the guanine (G) bases is essential. However, the contribution of each Hoogsteen base-pair at different positions to whole stability of the G-quadruplex has not been known. In this study, the effect of a deficiency of the Hoogsteen type hydrogen bond in the G-quadruplex stability was investigated. Spectral properties of meso-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP) associated with various G-quadruplexes were also examined.MethodsThe thermal stability of the thrombin-binding DNA aptamer 5′G₁G₂TTG₅G₆TG₈TG₁₀G₁₁TTG₁₄G₁₅ G-quadruplex, in which the guanine (G) base at 1, 2, 5, 6 and 8th positions was replaced with an inosine (I) base, one at a time, was investigated by circular dichroism (CD). The absorption, CD and fluorescence decay curve for the G-quadruplex associated TMPyP were also measured.ResultsThe transition from the G-quadruplex to a single stranded form was endothermic and induced by an increase in entropy. The order in stability was 0>8>6>2>5>1, where the numbers denote the position of the replacement and 0 represents no replacements of the G base, suggesting the significant contribution of the G₁ base in the stability of the G-quadruplex. Alteration in the spectral property of TMPyP briefly followed the order in thermal stability.ConclusionsReplacement of a G base with an I base resulted in destabilization of the G-quadruplex. The missing hydrogen bond at position 1 destabilized the G-quadruplex most efficiently. TMPyP binds near the I base-replaced location namely, the side of the G-quadruplex.General significanceThe Hoogsteen base-pairing is confirmed to be essential in stabilization of G-quadruplex. When G is replaced with I, the latter base is mobile to interact with cationic porphyrin.     

Preferential binding of a G-quadruplex ligand to human chromosome ends

The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, ³H-360A (2,6-N,N′-methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-³H]. We verified the in vitro selectivity of ³H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that ³H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3′-overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with ³H-360A. We found that ³H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.     

Enantioselective light switch effect of Δ- and Λ-[Ru(phenanthroline)2 dipyrido[3,2-a:2′, 3′-c]phenazine]2+ bound to G-quadruplex DNA

The interaction of Δ- and Λ-[Ru(phen)₂DPPZ]²⁺ (DPPZ = dipyrido[3,2-a:2′, 3′-c]phenazine, phen = phenanthroline) with a G-quadruplex formed from 5′-G₂T₂G₂TGTG₂T₂G_2–3′(15-mer) was investigated. The well-known enhancement of luminescence intensity (the ‘light-switch’ effect) was observed for the [Ru(phen)₂DPPZ]²⁺ complexes upon formation of an adduct with the G-quadruplex. The emission intensity of the G-quadruplex-bound Λ-isomer was 3-fold larger than that of the Δ-isomer when bound to the G-quadruplex, which is opposite of the result observed in the case of double stranded DNA (dsDNA); the light switch effect is larger for the dsDNA-bound Δ-isomer. In the job plot of the G-quadruplex with Δ- and Λ-[Ru(phen)₂DPPZ]²⁺, a major inflection point for the two isomers was observed at x ≈ .65, which suggests a binding stoichiometry of 2:1 for both enantiomers. When the G base at the 8th position was replaced with 6-methyl isoxanthopterin (6MI), a fluorescent guanine analog, the excited energy of 6-MI transferred to bound Δ- or Λ-[Ru(phen)₂DPPZ]²⁺, which suggests that at least a part of both Ru(II) enantiomers is close to or in contact with the diagonal loop of the G-quadruplex. A luminescence quenching experiment using [Fe(CN)₆]^4- for the G-quadruplex-bound Ru(II) complex revealed downward bending curves for both enantiomers in the Stern–Volmer plot, which suggests the presence of Ru(II) complexes that are both accessible and inaccessible to the quencher and may be related to the 2:1 binding stoichiometry.     

Emulsified BMVC derivative induced filtration for G-quadruplex DNA structural separation

A novel method based on emulsion/filtration is introduced for G-quadruplex DNA structural separation. We first synthesized a lipophilic analogue of BMVC, 3,6-Bis(1-methyl-4-vinylpyridinium)-9-(12′-bromododecyl) carbazole diiodide (BMVC-12C-Br), which can form an oil-in-water (o/w) phase emulsion. Due to the binding preferences of BMVC-12C-Br emulsion to some specific DNA structures, the large emulsion (∼2 µm) bound DNA was separated from the small free DNA in the filtrate by a 0.22 µm pore size MCE membrane. This method is able to isolate the non-parallel G-quadruplexes from the parallel G-quadruplexes and the linear duplexes from both G-quadruplexes. In addition, this method allows us not only to determine the absence of the parallel G-quadruplexes of d(T₂AG₃)₄ and the presence of the parallel G-quadruplexes of d(T₂AG₃)₂ in K⁺ solution, but also to verify structural conversion from antiparallel to parallel G-quadruplexes of d[AG₃(T₂AG₃)₃] in K⁺ solution under molecular PEG condition. Moreover, this emulsion can separate the non-parallel G-quadruplexes of d(G₃CGCG₃AGGAAG₅CG₃) monomer from the parallel G-quadruplexes of its dimer in K⁺ solution. Together with NMR spectra, one can simplify the spectra for both the free DNA and the bound DNA to establish a spectrum-structure correlation for further structural analysis.     

Benzo(h)quinoline derivatives as G-quadruplex binding agents

G-quadruplexes are unusual structures formed from guanine-rich sequences of nucleic acids. G-quadruplexes have been postulated to play important roles in a number of biological systems including gene regulation and the inhibition of enzyme function. Recently, our laboratory reported on the synthesis and evaluation of a triaza-cyclopentaphenanthrene compound which bound to G-quadruplexes with good affinity and selectivity. This compound contains a 4-pyridone group which has not been previously utilized in other quadruplex binding agents. In this Letter, we describe the synthesis and evaluation of 4-pyridone containing 2- and 3-carboxy-benzoquinolines as G-quadruplex binding agents. We find that these compounds are capable of binding G-quadruplexes with a K_a in the range of 3 × 10⁵ M^?1 and with a 10-fold selectivity for quadruplex over duplex DNA.     

Strand directionality affects cation binding and movement within tetramolecular G-quadruplexes

Nuclear magnetic resonance study of G-quadruplex structures formed by d(TG₃T) and its modified analogs containing a 5′-5′ or 3′-3′ inversion of polarity sites, namely d(3′TG5′-5′G₂T3′), d(3′T5′-5′G₃T3′) and d(5′TG3′-3′G₂T5’) demonstrates formation of G-quadruplex structures with tetrameric topology and distinct cation-binding preferences. All oligonucleotides are able to form quadruplex structures with two binding sites, although the modified oligonucleotides also form, in variable amounts, quadruplex structures with only one bound cation. The inter-quartet cavities at the inversion of polarity sites bind ammonium ions less tightly than a naturally occurring 5′-3′ backbone. Exchange of ¹⁵ ions between G-quadruplex and bulk solution is faster at the 3′-end in comparison to the 5′-end. In addition to strand directionality, cation movement is influenced by formation of an all-syn G-quartet. Formation of such quartet has been observed also for the parent d(TG₃T) that besides the canonical quadruplex with only all-anti G-quartets, forms a tetramolecular parallel quadruplex containing one all-syn G-quartet, never observed before in unmodified quadruplex structures.     

4-(1H-Imidazo[4,5-f]-1,10-phenanthrolin-2-yl)phenol-based G-quadruplex DNA binding agents: Telomerase inhibition,cytotoxicity and DNA-binding studies

Four novel 4-(1H-imidazo[4,5-f]-1,10-phenanthrolin-2-yl)phenol derivatives 1–4 have been synthesized, and their G-quadruplex DNA-binding interactions, telomerase inhibition, antiproliferative activity, cell cycle arrest, and apoptotic induction were studied. All compounds show the preferential h-telo, c-myc, and c-kit2 G-quadruplex binding affinity and the G-quadruplex versus duplex selectivity. In the case of the same G-quadruplex target, the compound 1 exhibits better stabilization effect (ΔT_m) than the other three compounds and also gives 80.2% inhibition of telomerase activity at 7.5 μM. All compounds can promote selectively the formation of parallel G-quadruplex structure of both c-myc and c-kit2 without addition of any cations. Four compounds display the cytotoxicity activities against HeLa and HepG2 cells by MTT assay with IC₅₀ values of about 10^?6 and 10^?5 M, respectively, and cause a substantial decrease in the G₂/M-phase cell population and a significant increase in the number of apoptotic cells.     

The 3D structures of G-Quadruplexes of HIV-1 integrase inhibitors: molecular dynamics simulations in aqueous solution and in the gas phase

The unimolecular G-quadruplex structures of d(GGGTGGGTGGGTGGGT) (G1) and d(GTGGTGGGTGGGTGGGT) (G2) are known as the potent nanomolar HIV-1 integrase inhibitors, thus investigating the 3D structures of the two sequences is significant for structure-based rational anti-HIV drug design. In this research, based on the experimental data of circular dichroism (CD) spectropolarimetry and electrospray ionization mass spectrometry (ESI-MS), the initial models of G1 and G2 were constructed by molecular modeling method. The modeling structures of G1 and G2 are intramolecular parallel-stranded quadruplex conformation with three guanine tetrads. Particularly, the structure of G2 possesses a T loop residue between the first and the second G residues that are the component of two adjacent same-stranded G-tetrad planes. This structure proposed by us has a very novel geometry and is different from all reported G-quadruplexes. The extended (35 ns) molecular dynamic (MD) simulations for the models indicate that the G-quadruplexes maintain their structures very well in aqueous solution whether the existence of K⁺ or NH₄⁺ in the central channel. Furthermore, we perform 500 ns MD simulations for the models in the gas phase. The results show that all the ion-G-quadruplex complexes are maintained during the whole simulations, despite the large magnitude of phosphate-phosphate repulsions. The gas phase MD simulations provide a good explanation to ESI-MS experiments. Our 3D structures for G1 and G2 will assist in understanding geometric formalism of G-quadruplex folding and may be helpful as a platform for rational anti-HIV drug design.     

Improving the affinity of naphthalene diimide ligand to telomeric DNA by incorporating Zn2+ ions into its dipicolylamine groups

N,N′-bis[3-[3-(2,2′-dipicolyl)methylaminopropyl]-methylaminopropyl]naphthalene-1,4,5,8-tetracarboxylic acid diimide, 1, and its complex with zinc ions, 2, were investigated against telomeric sequences, [TAGGG(TTAGGG)₃] and [AGGG(TTAGGG)₃], which reveal different G-quadruplex structures depending on the conditions. Spectrophotometric, SPR, and CD techniques revealed that both ligands showed large binding constants to hybrid-type G-quadruplexes formed in the presence of K⁺ ions. Moreover, 2 revealed higher affinity to investigated oligonucleotides suggesting that complex of naphthalene diimide derivative with Zn²⁺, comparing to 1, provided additional electrostatic or coordination interactions between positively charged zinc ions and condensed negative charged phosphate anions from G4 DNA.     

Human telomere, oncogenic promoter and 5'-UTR G-quadruplexes: diverse higher order DNA and RNA targets for cancer therapeutics

Guanine-rich DNA sequences can form G-quadruplexes stabilized by stacked G–G–G–G tetrads in monovalent cation-containing solution. The length and number of individual G-tracts and the length and sequence context of linker residues define the diverse topologies adopted by G-quadruplexes. The review highlights recent solution NMR-based G-quadruplex structures formed by the four-repeat human telomere in K⁺ solution and the guanine-rich strands of c-myc, c-kit and variant bcl-2 oncogenic promoters, as well as a bimolecular G-quadruplex that targets HIV-1 integrase. Such structure determinations have helped to identify unanticipated scaffolds such as interlocked G-quadruplexes, as well as novel topologies represented by double-chain-reversal and V-shaped loops, triads, mixed tetrads, adenine-mediated pentads and hexads and snap-back G-tetrad alignments. The review also highlights the recent identification of guanine-rich sequences positioned adjacent to translation start sites in 5′-untranslated regions (5′-UTRs) of RNA oncogenic sequences. The activity of the enzyme telomerase, which maintains telomere length, can be negatively regulated through G-quadruplex formation at telomeric ends. The review evaluates progress related to ongoing efforts to identify small molecule drugs that bind and stabilize distinct G-quadruplex scaffolds associated with telomeric and oncogenic sequences, and outlines progress towards identifying recognition principles based on several X-ray-based structures of ligand–G-quadruplex complexes.     

Disordering of human telomeric G-quadruplex with novel antiproliferative anthrathiophenedione

Linear heteroareneanthracenediones have been shown to interfere with DNA functions, thereby causing death of human tumor cells and their drug resistant counterparts. Here we report the interaction of our novel antiproliferative agent 4,11-bis[(2-{[acetimido]amino}ethyl)amino]anthra[2,3-b]thiophene-5,10-dione with telomeric DNA structures studied by isothermal titration calorimetry, circular dichroism and UV absorption spectroscopy. New compound demonstrated a high affinity (K_ass∼10⁶ M⁻¹) for human telomeric antiparallel quadruplex d(TTAGGG)₄ and duplex d(TTAGGG)₄∶d(CCCTAA)₄. Importantly, a ∼100-fold higher affinity was determined for the ligand binding to an unordered oligonucleotide d(TTAGGG TTAGAG TTAGGG TTAGGG unable to form quadruplex structures. Moreover, in the presence of Na⁺ the compound caused dramatic conformational perturbation of the telomeric G-quadruplex, namely, almost complete disordering of G-quartets. Disorganization of a portion of G-quartets in the presence of K⁺ was also detected. Molecular dynamics simulations were performed to illustrate how the binding of one molecule of the ligand might disrupt the G-quartet adjacent to the diagonal loop of telomeric G-quadruplex. Our results provide evidence for a non-trivial mode of alteration of G-quadruplex structure by tentative antiproliferative drugs.     

Evidence of different G-quadruplex DNA binding with biogenic polyamines probed by electrospray ionization-quadrupole time of flight mass spectrometry,circular dichroism and atomic force microscopy

The binding properties of five G-quadruplex oligonucleotides (humtel24, k-ras32, c-myc22, c-kit1 and c-kit2) with polyamines have been investigated by electrospray ionization-quadrupole time of flight mass spectrometry, circular dichroism, melting temperature, atomic force microscopy (AFM) and molecular simulation. The MS results demonstrated that the polyamines and G-quadruplex DNA can form complexes with high affinity, and one molecule of G-quadruplex DNA can combine several molecules (1–5) of polyamines. The binding affinities of the polyamines to DNA were in the order of spermine > spermidine > putrescine. After binding with polyamines, the conformations of the G-quadruplex DNA were significantly changed, and spermine can induce the configurations of k-ras32 and c-kit1 to deviate from their G-quadruplex structures at high concentrations. In the presence of K⁺, the conformations of G-quadruplex DNA were stabilized, while polyamines can also induced alterations of their configurations. Melting temperature experiments suggested that the T_m of the DNA–polyamine complexes obviously increased both in the absence and presence of K⁺. The AFM results indicated that polyamines can induce aggregation of G-quadruplex DNA. Above results illustrated that the polyamines bound with the phosphate backbone and the base-pairs of G-quadruplex structures. Combining with the molecular simulation, the binding mode of the G-quadruplex DNA and polyamines were discussed. The results obtained would be beneficial for understanding the biological and physiological functions of polyamines and provide useful information for development of antitumor drugs.     

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and ³¹P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (ΔH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were − 18.1 kcal/mol and − 15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K_p) was found to be 3.9 × 10³ M^− 1. ³¹P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. ³¹P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, ³¹P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.