Rotational Echo Double Resonance Experiments Research Articles

Solid-state NMR spectroscopic studies of 13C,15N,29Si-enriched biosilica from the marine diatom Cyclotella cryptica

Diatoms are algae producing micro- and nano-structured cell walls mainly containing amorphous silica. The shape and patterning of these cell walls is species-specific. Herein, the biosilica of Cyclotella cryptica, a centric marine diatom with a massive organic matrix, is studied. Solid-state NMR spectroscopy is applied to gain deeper insight into the interactions at the organic–inorganic interface of the cell walls. The various organic compounds like polysaccharides as well as proteins and long-chain polyamines (LCPAs) are detected by observation of heteronuclei like 13C and 15N whereas the silica phase is studied using 29Si NMR spectroscopy. The sensitivity of the NMR experiments is strongly enhanced by isotope-labeling of the diatoms during cultivation with 13C, 15N and 29Si. The presence of two different chitin species in the biosilica is demonstrated. This observation is supported by a monosaccharide analysis of the silica-associated organic matrix where a high amount of glucosamine is found. Moreover, the Rotational Echo Double Resonance (REDOR) experiment provides distance information for heteronuclear spins. 13C{29Si} REDOR experiments reveal proximities between different organic compounds and the silica phase. The closest contacts between silica and organic compounds appear for different signals in the 13C-chemical shift range of 40–60 ppm, the typical range for LCPAs.

Lamellar Structure in Alanine-Glycine Copolypeptides Studied by Solid-State NMR Spectroscopy: A Model for the Crystalline Domain of Bombyx mori Silk Fibroin in Silk II Form.

Bombyx mori silk fibroin (SF) fibers with excellent mechanical properties have attracted widespread attention as new biomaterials. However, the structural details are still not conclusive. Here, we propose a lamellar structure for the crystalline domain of the SF fiber based on structural analyses of the Ala Cβ peaks in the 13C cross-polarization/magic angle spinning NMR spectra of (Ala-Gly)m (m = 9, 12, 15, and 25) and 13C selectively labeled (Ala-Gly)15 model peptides. Namely, three Ala Cβ peaks with relative intensities of 1:2:1 obtained by deconvolution were assigned to two kinds of β-sheet and a β-turn, which are interpreted as a lamellar structure formed by repetitive folding using β-turns every eighth amino acid, for which the basic structure is (Ala-Gly)4 in an antipolar arrangement. The dynamics and intermolecular arrangement were further studied using 13C solid-state spin-lattice relaxation time observations and the rotational echo double resonance experiments, respectively.

1H, 29Si and 119Sn double and triple resonance NMR spectroscopy of the small-pore framework sodium stannosilicate Na2SnSi3O9·2H2O

The small-pore framework sodium stannosilicate AV-10, chemical composition Na2SnSi3O9·2H2O and known crystallographic structure, was synthesized by hydrothermal crystallization. This stannosilicate is built up of a three-dimensional network of corner-shared SiO4 tetrahedra and SnO6 octahedra. The SnO6 sites are linked to six SiO4 tetrahedra (Sn(6Si)) while each of the two crystallographically different SiO4 units are connected to two SnO6 and SiO4 units (Si(2Si,2Sn)). This material was used as model compound for developing a solid-state MAS NMR strategy aimed on the challenges and possibilities for structural studies, particularly considering the short and medium range order to verify the connectivity of SiO4 and SnO6 of such compounds despite the low natural abundances of 4.68% for 29Si and 8.59% for 119Sn nuclei as a real challenge. 29Si{119Sn} and 119Sn{29Si} REDOR (Rotational-Echo Double-Resonance) NMR measurements after 1H cross-polarization (CP) were carried out. The REDOR curves show a significant change after the “normal” quadratic short time evolution from which both (i) the shortest internuclear 29Si – 119Sn distances (and vice versa) and (ii) the number of corner-sharing SiO4 tetrahedra around the SnO6 octahedra (and vice versa) can be obtained. Based on these data, optimized 29Si{119Sn} and 119Sn{29Si} REPT-HMQC (Recoupled Polarization Transfer-Heteronuclear Multiple-Quantum Correlation, again after 1H CP) experiments were implemented, which directly show those heterogroup connectivity as correlation peaks in a 2D spectrum. This information was also obtained using 2D29Si{119Sn}-J-Coupling NMR experiments. Furthermore, 2D29Si INADEQUATE NMR experiments are also feasible, showing the connectivity of SiO4 tetrahedra. The combination of REDOR, REPT-HMQC, J-Coupling and INADEQUATE experiments yielded a complete analysis of the short and medium range structure of this microporous stannosilicate, in agreement with the previously published structure obtained Ab Initio from powder X-Ray diffraction data (XRD).

Overcoming Prohibitively Large Radiofrequency Demands for the Measurement of Internuclear Distances with Solid-State NMR under Fast Magic-Angle Spinning.

Solid-state NMR is a powerful tool to measure distances and motional order parameters which are vital tools in characterizing the structure and dynamics of molecules. Magic-angle spinning (MAS), widely employed in solid-state NMR, averages out dipole-dipole couplings that carry such information. Hence, rotor-synchronized radiofrequency (RF) pulses, that interfere with MAS averaging, are commonly employed to measure such couplings. However, most of the methods that achieve this, rotational echo double resonance (REDOR) being a classic example, require RF amplitudes that are greater than or equal to the MAS frequency. While feasible at MAS frequencies <40 kHz, these requirements become prohibitively large for higher MAS frequencies (40-110 kHz), which are now commercially available. Here, we redesign the REDOR experiment so that RF amplitudes as low as 0.5-0.7 times the spinning frequency can be used. This sequence, name deferred rotational echo double resonance (DEDOR), thus extends the utility of this method to the fastest MAS frequencies currently commercially available (111 kHz). The generality of this strategy is shown by extending it to other methods that utilize the same principle as REDOR. They will be useful in obtaining structural parameters for a wide range of molecules using solid-state NMR under fast MAS with the additional advantage of higher spectral resolution under these conditions.

On the direct relation between REDOR and DIPSHIFT experiments in solid-state NMR.

Rotational-echo double resonance (REDOR) and Dipolar-coupling chemical-shift correlation (DIPSHIFT) are commonly used experiments to probe heteronuclear dipole-dipole couplings between isolated pairs of spin-12 nuclei in magic-angle-spinning (MAS) solid-state NMR. Their widespread use is due to their robustness to experimental imperfections and a straightforward interpretation of data. Both of these experiments use rotor-synchronised π pulses to recouple the heteronuclear dipole-dipole couplings, and the observed intensity of resonances is modulated by a recoupled phase factor depending on the position or duration of the recoupling pulses. Several modifications to both of these experiments have been proposed, for example, the development of DIPSHIFT which employs strategies that mimic the multi-rotor-period nature of REDOR. We show here that REDOR and DIPSHIFT are in fact alternate implementations of the same experiment. The overt similarity in the design of REDOR and DIPSHIFT is also reflected in their theoretical description. Dipolar dephasing curves in REDOR are obtained by increasing the recoupling duration whilst keeping the position of the pulses constant, which results in a dephasing factor that is a function of only the dephasing time. DIPSHIFT, on the other hand, is a constant-time version of REDOR; the dipolar dephasing is a function of the position of the pulses with respect to the rotor period. We discuss the advantages and disadvantages of each implementation and suggest domains of applicability for these sequences.

Structural Studies of NaPO3–AlF3 Glasses by High-Resolution Double-Resonance Nuclear Magnetic Resonance Spectroscopy

The local structure of the model glasses (NaPO3)1–x-(AlF3)x (0 ≤ x ≤ 0.4), prepared by standard melt-cooling, was extensively investigated by high-resolution solid-state nuclear magnetic resonance (NMR) including advanced double-resonance techniques. This glass system offers the opportunity of studying five different heteronuclear distance correlations (Na–F, Na–P, P–F, Al–F, and P–Al) by 10 distinct double-resonance experiments, involving all of the constituent elements present. 27Al MAS-NMR data indicate that aluminum is predominantly six-coordinated. According to 27Al{31P} and 27Al{19F} rotational-echo double-resonance (REDOR) spectroscopic results, two to three Al–F and three to four Al–O–P linkages occur in these glasses, independent of composition x. 19F MAS-NMR spectra show the presence of terminal P-bound and Al-bound fluorine species. A small amount of fluorine bridging to two aluminum octahedra, which could be assigned based on 19F{27Al} and 19F{31P} REDOR experiments, was also detected. 19F{23N...

Rapid measurement of long-range distances in proteins by multidimensional 13C-19F REDOR NMR under fast magic-angle spinning.

The ability to simultaneously measure many long-range distances is critical to efficient and accurate determination of protein structures by solid-state NMR (SSNMR). So far, the most common distance constraints for proteins are 13C-15N distances, which are usually measured using the rotational-echo double-resonance (REDOR) technique. However, these measurements are restricted to distances of up to ~ 5Å due to the low gyromagnetic ratios of 15N and 13C. Here we present a robust 2D 13C-19F REDOR experiment to measure multiple distances to ~ 10Å. The technique targets proteins that contain a small number of recombinantly or synthetically incorporated fluorines. The 13C-19F REDOR sequence is combined with 2D 13C-13C correlation to resolve multiple distances in highly 13C-labeled proteins. We show that, at the high magnetic fields which are important for obtaining well resolved 13C spectra, the deleterious effect of the large 19F chemical shift anisotropy for REDOR is ameliorated by fast magic-angle spinning and is further taken into account in numerical simulations. We demonstrate this 2D 13C-13C resolved 13C-19F REDOR technique on 13C, 15N-labeled GB1. A 5-19F-Trp tagged GB1 sample shows the extraction of distances to a single fluorine atom, while a 3-19F-Tyr labeled GB1 sample allows us to evaluate the effects of multi-spin coupling and statistical 19F labeling on distance measurement. Finally, we apply this 2D REDOR experiment to membrane-bound influenza B M2 transmembrane peptide, and show that the distance between the proton-selective histidine residue and the gating tryptophan residue differs from the distances in the solution NMR structure of detergent-bound BM2. This 2D 13C-19F REDOR technique should facilitate SSNMR-based protein structure determination by increasing the measurable distances to the ~ 10Å range.

Reprint of: Localization of Cl-35 Nuclei in Biological Solids using Rotational-Echo Double-Resonance Experiments

Chloride ions play important roles in many chemical and biological processes. This paper investigates the possibility of localizing 35Cl nuclei using solid-state NMR. It demonstrates that distances shorter than 3.8Å, between 13C atoms and 35Cl atoms in 10% uniformly labeled 13C L-tyrosine·HCl and natural abundance Glycine·HCl can be measured using rotational-echo (adiabatic passage) double-resonance (RE(AP)DOR). Furthermore the effect of quadrupolar interaction on the REDOR/REAPDOR experiment is quantified. The dephasing curve is plotted in a three dimensional chart as a function of the dephasing time and of the strength of quadrupolar interaction felt by each orientation. During spinning each orientation feels a quadrupolar interaction that varies in time, and therefore at each moment in time we reorder the crystallite orientations as a function of their contribution to the dephasing curve. In this way the effect of quadrupolar interaction on the dipolar dephasing curve can be fitted with a polynomial function. The numerical investigation performed allows us to generate REDOR/REAPDOR curves which are then used to simulate the experimental data.

Spectroscopic Characterization of Adsorbed 13CO2 on 3-Aminopropylsilyl-Modified SBA15 Mesoporous Silica.

Multiple chemisorption products are found from the interaction of CO2 with the solid-amine sorbent, 3-aminopropyl silane (APS), bound to mesoporous silica (SBA15) using solid-state NMR and FTIR spectroscopy. We employed a combination of both 15N{13C} rotational-echo double-resonance (REDOR) NMR and 13C{15N} REDOR to determine the chemical identity of these products. 15N{13C} REDOR measurements are consistent with a single 13C-15N pair and distance of 1.45 Å. In contrast, both 13C{15N} REDOR and 13C CPMAS are consistent with multiple 13C products. 13C CPMAS shows two neighboring resonances, whose chemical shifts are consistent with carbamate (at 165 ppm) and carbamic acid. The 13C{15N} REDOR experiments resonant at 165 ppm show an incomplete buildup of the REDOR data to ∼90% of the expected maximum. We conclude this 10% missing intensity corresponds to a 13C NMR species that resonates at the identical chemical shift but that is not in dipolar contact with 15N. These data are consistent with the presence of bicarbonate, HCO3-, since it is commonly observed at ∼165 ppm and lacks 15N for dipolar coupling.

Localization of Cl-35 nuclei in biological solids using rotational-echo double-resonance experiments

Chloride ions play important roles in many chemical and biological processes. This paper investigates the possibility of localizing 35Cl nuclei using solid-state NMR. It demonstrates that distances shorter than 3.8Å, between 13C atoms and 35Cl atoms in 10% uniformly labeled 13C L-tyrosine·HCl and natural abundance Glycine·HCl can be measured using rotational-echo (adiabatic passage) double-resonance (RE(AP)DOR). Furthermore the effect of quadrupolar interaction on the REDOR/REAPDOR experiment is quantified. The dephasing curve is plotted in a three dimensional chart as a function of the dephasing time and of the strength of quadrupolar interaction felt by each orientation. During spinning each orientation feels a quadrupolar interaction that varies in time, and therefore at each moment in time we reorder the crystallite orientations as a function of their contribution to the dephasing curve. In this way the effect of quadrupolar interaction on the dipolar dephasing curve can be fitted with a polynomial function. The numerical investigation performed allows us to generate REDOR/REAPDOR curves which are then used to simulate the experimental data.

The Structure of the Bimolecular Complex between Amphotericin B and Ergosterol in Membranes Is Stabilized by Face-to-Face van der Waals Interaction with Their Rigid Cyclic Cores.

Amphotericin B (AmB) is a polyene macrolide antibiotic isolated from Streptomyces nodosus. The antifungal activity of AmB can be attributed to the formation of an ion-channel assembly in the presence of ergosterol (Erg), in which there are two different AmB-Erg orientations, parallel and antiparallel, as reported previously. In this study, to elucidate the structures of those AmB-Erg complexes based on solid-state nuclear magnetic resonance, a (19)F-labeled AmB derivative was newly prepared by a hybrid synthesis that utilized degradation products from the drug. Using the 2-(trimethylsilyl)ethoxymethyl (SEM) group as the protecting group for the carboxylic acid moiety of AmB, the fully deprotected labeled AmB compounds were obtained successfully. Then, these labeled AmBs were subjected to (13)C{(19)F} rotational-echo double-resonance (REDOR) experiments in hydrated lipid bilayers. The results indicated the coexistence of parallel and antiparallel orientations for AmB and Erg pairing, at a ratio of 7:3. A total of six distances between AmB and Erg were successfully obtained. Geometry analysis using the distance constraints derived from the REDOR experiments provided the plausible AmB-Erg complex structure for both the parallel and antiparallel interactions. The flat macrolide of AmB and the tetracyclic core of Erg closely contacted in a face-to-face manner, thus maximizing the van der Waals interaction between the two molecules. This interaction can be attributed to the coexistence of both the parallel and antiparallel orientations.

An optimal double-magic flip angle for performing the distance measurement REDOR experiment on a spin S=1

Distance measurements between a half-spin and a quadrupolar S=1 spin having a small quadrupolar coupling constant can be performed using the rotational echo double resonance (REDOR) experiment. We derived an analytical expression for the probability of transitions between energy levels resulting from the application of an arbitrary pulse flip angle to the quadrupolar spin and consequently minimized the probability that populations of individual levels do not undergo a spin transition during the pulse. As a result we discovered that if the flip angle of the quadrupolar spin pulse is 109.47°, the maximal recoupling values are the largest possible and the signal reaches a maximum value of 8/9, larger than in the use of either a 90° pulse or a 180° pulse. In addition, the slope of the initial decay is higher than that of the 90° pulse. The recoupling signal can be modeled by an exact analytical formula in the ideal case and simulations show that the advantage of the 109.47° pulse is preserved when the quadrupolar coupling constant CQ has a finite value typical of 2H and 6Li spins (up to CQ~200kHz). Experimental results on two spin pairs, 2H–13C and 6Li–13C, demonstrate the validity and accuracy of this method.

Intermediate Role of Gallium in Oxidic Glasses: Solid State NMR Structural Studies of the Ga2O3–NaPO3 System

A series of (NaPO3)1–x(Ga2O3)x glasses (0 ≤ x ≤ 0.35) prepared by conventional melt-quenching methods has been structurally characterized by various complementary high resolution one-dimensional and two-dimensional (2D) solid state magic angle spinning nuclear magnetic resonance (MAS NMR) techniques, which were validated by corresponding experiments on the crystalline model compounds GaPO4 (quartz) and Ga(PO3)3. Alloying NaPO3 glass by Ga2O3 results in a marked increase in the glass transition temperature, similar to the effect observed with Al2O3. At the atomic level, multiple phosphate species QnmGa (n = 0, 1, and 2; m = 0, 1, 2, and 3) can be observed. Here n denotes the number of P–O–P and m the number of P–O–Ga linkages, and (m + n ≤ 4). For resolved resonances, the value of n can be quantified by 2D J-resolved spectroscopy, refocused INADEQUATE, and a recently developed homonuclear dipolar recoupling method termed DQ-DRENAR (double-quantum based dipolar recoupling effects nuclear alignment reduction). Ga3+ is dominantly found in six-coordination in low-Ga glasses, whereas in glasses with x > 0.15, lower-coordinated Ga environments are increasingly favored. The connectivity between P and Ga can be assessed by heteronuclear 71Ga/31P dipolar recoupling experiments using 71Ga{31P} rotational echo double resonance (REDOR) and 31P {71Ga} rotational echo adiabatic passage double resonance (READPOR) techniques. Up to x = 0.25, the limiting composition where this is possible, the second coordination sphere of all the gallium atoms is fully dominated by phosphorus atoms. Above x = 0.25, 71Ga static and MAS NMR as well as REDOR experiments give clear spectroscopic evidence of Ga–O–Ga connectivity. 31P/23Na REDOR and REAPDOR results indicate that gallium has no dispersion effect on sodium ions in these glasses. They also indicate significant differences in the strength of dipolar interactions for distinct QnmGa species, consistent with bond valence considerations. On the basis of these results, a comprehensive structural model is developed. This model explains the compositional trend of the glass transition temperatures in terms of the concentration of bridging oxygen species (P–O–P, P–O–Ga, and Ga–O–Ga) in these glasses. The results provide new insights into the role of Ga2O3 as an intermediate oxide, with features of both network modifier and network former in oxide glasses.

ChemInform Abstract: REDOR NMR for Drug Discovery

AbstractReview: 56 refs.

Fast REDOR with CPMG multiple-echo acquisition

Rotational-Echo Double Resonance (REDOR) is a widely used experiment for distance measurements in solids. The conventional REDOR experiment measures the signal dephasing from hetero-nuclear recoupling under magic-angle spinning (MAS) in a point by point manner. A modified Carr–Purcell Meiboom–Gill (CPMG) multiple-echo scheme is introduced for fast REDOR measurement. REDOR curves are measured from the CPMG echo amplitude modulation under dipolar recoupling. The real time CPMG–REDOR experiment can speed up the measurement by an order of magnitude. The effects from hetero-nuclear recoupling, the Bloch–Siegert shift and echo truncation to the signal acquisition are discussed and demonstrated.

Robust NMR Approaches for the Determination of Homonuclear Dipole–Dipole Coupling Constants in Studies of Solid Materials and Biomolecules

This review addresses the NMR spectroscopy study of molecular structure and dynamics by way of homonuclear dipole-dipole couplings by relying on their orientation and direct distance dependence. The study of homonuclear couplings as opposed to heteronuclear couplings poses specific challenges. On the one hand, two like spins cannot be independently manipulated easily, which means that simple shift-refocusing concepts by using hard π pulses cannot be used to cope with potentially large chemical-shift dispersions at the high fields used today. On the other hand, the noncommutativity of the different pair Hamiltonians in a multispin system leads to complications associated with the isolation of specific pair couplings while minimizing the influence of the other spins. In particular, the so-called dipolar-truncation effect challenges the observation of weak couplings of interest in the presence of stronger ones. Recent advances in determining homonuclear dipole-dipole coupling constants are reviewed, stressing the use of double-quantum spectroscopy approaches and their similarity to the popular heteronuclear rotational-echo double-resonance experiment. Particular emphasis is put on corrections for the influence of transverse relaxation effects on the measured data, and the handling of distribution effects as well as potential dynamic heterogeneities in complex substances.

Insights into the spin dynamics of a large anisotropy spin subjected to long-pulse irradiation under a modified REDOR experiment

Distance measurements between a spin-1/2 and a second spin bearing a large anisotropy are performed using a modified rotational echo double resonance (REDOR) experiment. By applying pairs of rotor-synchronized π pulses on the detected spin and a single long pulse on the coupled spin the dipolar interaction is efficiently recoupled even at the sudden passage limit where both adiabaticity and the hard pulse approximation are not valid. In this manuscript we derive the theoretical basis for analyzing the behavior of single crystallites in order to gain insight into the mechanism of dipolar recoupling, and in order to find conditions for optimizing the experiment. The use of reduced time and frequency variables show that the signal depends on the ratios of the radio frequency strength ν1 and the anisotropy, either the CSA (νσ) or the quadrupolar interaction (νQ), with respect to the spinning frequency νR. We derive expressions for the contribution of individual crystallites to the signal arising from the different frequencies mνd (m=0,1…2S) associated with the dipolar interaction and show that they result in a non-random distribution of intensities. For a spin-1/2 with a large CSA (up to 1MHz and more) we show using calculations and simulations that the result is a recoupling signal that takes maximal values ΔS/S0 of ∼0.6–0.7, beyond the saturation limit of 0.5, defined by equal contribution of all transitions. For a spin-3/2 we show that at certain conditions the non-random scrambling may result in an apparent saturation-like behavior. In all cases large RF amplitudes are not necessarily required for obtaining efficient recoupling. 13C–11B LA-REDOR (Low-Alpha/Low-rf-Amplitude REDOR) dipolar recoupling experiments on 4-methoxyphenylboronic acid were performed following optimization of the spinning rates suitable for low amplitude radio-frequency power levels and show that efficient recoupling can be obtained for a spin-3/2, and that distance determination is not very strongly dependent on the actual value of the quadrupolar coupling constant.

Possible conformation of amphotericin B dimer in membrane-bound assembly as deduced from solid-state NMR

Aiming for structural analysis of amphotericin B (AmB) ion-channel assemblies in membrane, a covalent dimer was synthesized between 13C-labled AmB methyl ester and 19F-labled AmB. The dimer showed slightly weaker but significant biological activities against fungi and red blood cells compared with those of monomeric AmB. Then the dimer was subjected to 13C{19F}REDOR (Rotational-Echo Double Resonance) experiments in hydrated lipid bilayers. The obtained REDOR dephasing effects were explained by two components; a short 13C/19F distance (6.9Å) accounting for 23% of the REDOR dephasing, and a longer one (14Å) comprising the rest of the dephasing. The shorter distance is likely to reflect the formation of barrel-stave ion channel.

Carbon–Deuterium Rotational-Echo Double-Resonance NMR Spectroscopy of Lyophilized Aspartame Formulations

In this study, changes in the local conformation of aspartame were observed in annealed lyophilized glasses by monitoring changes in the distance between two labeled sites using C-(2)H rotational-echo double-resonance (REDOR) nuclear magnetic resonance (NMR) spectroscopy. Confirmation that the REDOR experiments were producing accurate distance measurement was ensured by measuring the (13)C-(15)N distance in glycine. The experiment was further verified by measuring the REDOR dephasing curve on (13)C-(2)H methionine. (13)C-(2)H REDOR dephasing curves were then measured on lyophilized aspartame-disaccharide formulations. In aspartame-sucrose formulation, the internuclear distances increased upon annealing, which correlated with decreased chemical reactivity. By contrast, annealing had only a minimal effect on the dephasing curve in aspartame-trehalose formulation. The results show that stability is a function of both mobility and local structure (conformation), even in a small molecule system such as lyophilized aspartame-sucrose.

Head-to-Tail Interaction between Amphotericin B and Ergosterol Occurs in Hydrated Phospholipid Membrane

Amphotericin B (AmB) is thought to exert its antifungal activity by forming an ion-channel assembly in the presence of ergosterol. In the present study we aimed to elucidate the mode of molecular interactions between AmB and ergosterol in hydrated phospholipid bilayers using the rotational echo double resonance (REDOR) spectra. We first performed (13)C{(19)F}REDOR experiments with C14-(19)F-labeled AmB and biosynthetically (13)C-labeled ergosterol and implied that both "head-to-head" and "head-to-tail" orientations occur for AmB-ergosterol interaction in the bilayers. To further confirm the "head-to-tail" pairing, (13)C-labeled ergosterol at the dimethyl terminus (C26/C27) was synthesized and subjected to the REDOR measurements. The spectra unambiguously demonstrated the presence of a "head-to-tail" orientation for AmB-ergosterol pairing. In order to obtain information on the position of the dimethyl terminus of ergosterol in membrane, (13)C{(31)P}REDOR were carried out using the labeled ergosterol and the phosphorus atom of a POPC headgroup. Significant REDOR dephasing was observed at the C26/C27 signal of ergosterol in the presence of AmB, but not in the absence of AmB, clearly indicating that the side-chain terminus of ergosterol in the AmB complex comes close to the bilayer surface.