High-resolution 2D NMR spectroscopy of bicelles to measure the membrane interaction of ligands

Magnetically aligned bicelles are increasingly being used as model membranes in solution- and solid-state NMR studies of the structure, dynamics, topology, and interaction of membrane-associated peptides and proteins. These studies commonly utilize the PISEMA pulse sequence to measure dipolar coupling and chemical shift, the two key parameters used in subsequent structural analysis. In the present study, we demonstrate that the PISEMA and other rotating-frame pulse sequences are not suitable for the measurement of long-range heteronuclear dipolar couplings, and that they provide inaccurate values when multiple protons are coupled to a 13C nucleus. Furthermore, we demonstrate that a laboratory-frame separated-local-field experiment is capable of overcoming these difficulties in magnetically aligned bicelles. An extension of this approach to accurately measure 13C-31P and 1H-31P couplings from phospholipids, which are useful to understand the interaction of molecules with the membrane, is also described. In these 2D experiments, natural abundance 13C was observed from bicelles containing DMPC and DHPC lipid molecules. As a first application, these solid-state NMR approaches were utilized to probe the membrane interaction of an antidepressant molecule, desipramine, and its location in the membrane.     

Bilayer in small bicelles revealed by lipid-protein interactions using NMR spectroscopy

Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and small bicelles of dihexanoyl phosphatidylcholine (DHPC) and dimyristoyl phosphatidylcholine (DMPC) give insights into protein-lipid interactions. Intermolecular NOEs between hydrophobic tails of lipid and protein in the bicelles cover the surface area of OmpX forming a continuous cylindric jacket of approximately 2.7 nm in height. These NOEs originate only from DMPC molecules, and no NOEs from DHPC are observed. Further, these NOEs are mainly from methylene groups of the hydrophobic tails of DMPC, and only a handful of NOEs arise from methyl groups of the hydrophobic tails. The observed contacts indicate that the hydrophobic tails of DMPC are oriented parallel to the surface of OmpX and thus DMPC molecules form a bilayer in the vicinity of the protein. Thus, a bilayer exists in the small bicelles not only in the absence of but also in the presence of a membrane protein. In addition, the number of NOEs between the polar head groups of lipid molecules and protein is increased in the bicelles compared with those in micelles. This observation may be due to the closely packed head groups of the bilayer. Moreover, irregularity of hydrophobic interactions in the middle of the bilayer environment was observed. This observation together with the interactions between polar head groups and proteins gives a possible rationale for structural and functional differences of membrane proteins solubilized in micelles and in bilayer systems and hints at structural differences between protein-free and protein-loaded bilayers.     

Evidence of solvent-gelator interaction in sugar-based organogel studied by field-cycling NMR relaxometry

The dynamics of bulk toluene and toluene confined in the 1,2-O-(1-ethylpropylidene)-α-D-glucofuranose gel was studied using (1)H field-cycling nuclear magnetic resonance relaxometry. The proton spin-lattice relaxation time T(1) was measured as a function of the magnetic field strength and temperature. The observed dispersion in the frequency range 10(4)-10(6) Hz for the relaxation rate of toluene in the gel system give evidence of the interaction between the toluene and the gelator aggregates. The data were interpreted in terms of the two-fraction fast-exchange model. Additionally it was also shown that a cooling rate during gel preparation process influences the gel microstructure and leads to different gelator-solvent interactions as reflected in a different behavior of the proton spin-lattice relaxation rate of toluene within the gel observed at the low frequency range.     

Zeolite-included molecules studied by NMR

The investigation of xenon and large aromatic molecules adsorbed in zeolitic materials using NMR spectroscopy is discussed. The results suggest a system in which the molecules are often exchanging among adsorption sites. Careful analysis of the spectroscopic results allow one to determine parameters of the magnetic properties of the adsorption sites, as well as factors affecting the exchange.     

High-resolution NMR spectroscopy of a GPCR in aligned bicelles

Solid-state NMR spectra with single-site resolution of CXCR1, a G protein-coupled receptor (GPCR), were obtained in magnetically aligned phospholipid bicelles. These results demonstrate that GPCRs in phospholipid bilayers are suitable samples for structure determination by solid-state NMR. The spectra also enable studies of drug-receptor interactions.     

High-resolution NMR spectroscopy of membrane proteins in aligned bicelles

High-resolution solid-state NMR spectra can be obtained from uniformly (15)N-labeled membrane proteins in magnetically aligned bicelles. Fast uniaxial diffusion about the axis of the bilayer normal results in single-line spectra that contain the orientational information necessary for protein structure determination.     

Furanized rubber studied by NMR spectroscopy

The double bonds of natural rubber latex (stabilized by a nonionic surfactant) were reacted with an approximately equimolar amount of performic acid at room temperature with a limited amount of formic acid present. Product analysis by ¹H-NMR during the course of the reaction showed that 69–90% epoxidation occurred before the advent of ring opening and ring expansion to produce furanized rubber; hence the rate of epoxidation was greater than the rate of furanization. Indeed, at lower concentrations of formic acid and rubber latex, epoxidation occurred to 90% and furanization was prevented; it was subsequently brought about by the addition of a catalytic amount of orthophosphoric acid. Increased formic acid concentration caused early coagulation of the modified rubber latex. By ¹H- and ¹³C-NMR, it was found that the furanized rubber probably consisted of tetrahydrofuran rings linked together by C? C bonds at positions adjacent to the hetero atom and contained a terminal hydroxy group. The number average sequence length was 2–9, but only the sample with an average sequence length of 9 was effective as a cation binder.     

A high-resolution solid-state NMR approach for the structural studies of bicelles

Bicelles are increasingly being used as membrane mimicking systems in NMR experiments to investigate the structure of membrane proteins. In this study, we demonstrate the effectiveness of a 2D solid-state NMR approach that can be used to measure the structural constraints, such as heteronuclear dipolar couplings between 1H, 13C, and 31P nuclei, in bicelles without the need for isotopic enrichment. This method does not require a high radio frequency power unlike the presently used rotating-frame separated-local-field (SLF) techniques, such as PISEMA. In addition, multiple dipolar couplings can be measured accurately, and the presence of a strong dipolar coupling does not suppress the weak couplings. High-resolution spectra obtained from magnetically aligned DMPC:DHPC bicelles even in the presence of peptides suggest that this approach will be useful in understanding lipid-protein interactions that play a vital role in shaping up the function of membrane proteins.     

Membrane-mediated capillary electrophoresis: interaction of cationic peptides with bicelles

Electrokinetic capillary chromatography is applied to determine the membrane affinity of peptides using both 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles and DHPC/1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bicelles under controlled conditions. The effect of temperature and the bicelle q value in surface association with cationic peptides is studied. The cationic peptides selected have a well-defined membrane structure (indolicidin), induced secondary structure (melittin, magainin 2), or do not possess classical secondary structure (atrial natriuretic peptide (ANP) 1-28, 4-28, 5-27). Electrokinetic capillary chromatography facilitated by DMPC and DHPC additives provides a rapid means of estimating lipophilicity and screening for peptides that have membrane affinity.     

Polycondensation behavior of methyltrimethoxysilane studied by NMR spectroscopy

Silicate gels are most often synthesized by hydrolyzing monomeric alkoxide precursors employing acid or base catalysts. The properties of final products are determined by the pathway of the reactions, which includes hydrolysis, condensation, and solvent exchange reaction. We analyzed a system with methyltrimethoxysilane as monomer and ethanol as solvent by using ¹H, ¹³C, and 2D heteronuclear correlated nuclear magnetic resonance (NMR) spectroscopy. Complete assignments of the proton and the ¹³C-NMR bands were established, which rendered the kinetic study feasible. An understanding of the behavior of the system was also attempted, especially at earlier stages. The results showed that hydrolysis and solvent exchange reactions occurred at early stage whereas condensation occurred slowly. © 1996 John Wiley & Sons, Inc.     

Hydrogen exchange in benzamidoximes studied by NMR spectroscopy

The hydrogen exchange process in benzamidoximes ( 1 and 2 ) was studied over a range of temperature and a determination of the activation parameter ΔG? for this process was made.     

Structures of branched oligophenylenes studied by NMR spectroscopy

A series of model cyclotrimers was studied by NMR spectroscopy using 2D COSY, HSQC, and HMBC correlations to establish the structures of branched oligophenylenes, whose branching center is the 1,3,5-triphenyl-substituted benzene ring.     

Hydration of choline salts studied by NMR relaxation

The concentration dependence of T₁ for protons and deuterons in H₂O and D₂O solutions of three choline salts is anomalously high in dilute solutions. We suggest that this is due to formation of clathrate hydrate shells. Acetylcholine chloride does not show anomalous behaviour.     

Conformation and location of membrane-bound salinomycin-sodium complex deduced from NMR in isotropic bicelles

An ionophore antibiotic salinomycin was studied in a membrane environment consisting of isotropic bicelles, a better model for biological membranes than micelles, and its conformation and topological orientation in bicelles was determined. 2D NMR measurements and restrained conformational search revealed that salinomycin-sodium salt in bicelles adopts an open conformation in which the orientation of the E-ring is significantly different from that in crystal and solution structures. This conformational alteration breaks an intramolecular hydrogen bond between 28-OH and 1-O, dislocates the ether oxygen of the E-ring from a coordinated position to the sodium ion observed in the crystal, and consequently weakens the complexation between salinomycin and the sodium ion. Paramagnetic relaxation experiments using doxyl-phospholipids reveal that salinomycin is embedded shallowly in bicelles, with both terminals being closer to the water interface and the olefin portion facing the bicelle interior. Measurements of intermolecular NOEs between salinomycin and phospholipids further supported this orientation. Weaker complexation with sodium ion and positional preference in the membrane polar region may facilitate the catch-and-release of metal ions at the polar/nonpolar interface of bilayers. On the basis of these findings, a model for salinomycin-assisted transport of metal ions across biological membranes is proposed.     

Aggregation behavior of sodium deoxycholate and its interaction with cetyltrimethylammonium bromide in aqueous solution studied by NMR spectroscopy

In pure sodium deoxycholate (NaDC) dilute solution, the transverse relaxation times (T ₂) of nearly all proton signals of NaDC obey single-exponential decay, with exception for proton at 3-position (H3) that decays in a two-exponential manner. The two components with different mobility of H3 of NaDC indicate that the molecules are probably associated in head-to-tail pairs via hydrogen bonds in dilute solution. In the mixed NaDC/cetyltrimethylammonium bromide (CTAB) solutions, the T ₂ values of interested NaDC protons are far less than those in pure NaDC solution, especially when the concentrations of the two components are close to equal-molar. The results of self-diffusion coefficients and the chemical shifts confirm further that the strong interaction occurs between the two components in mixed solutions, especially for equal-molar condition. The arrangement of the mixed aggregates can be speculated for the cross-peaks of proton pairs occurring between NaDC and CTAB molecules in two-dimensional and rotating frame nuclear Overhauser enhancement spectroscopy spectra.     

Mobility of solid tert-butyl alcohol studied by deuterium NMR

The molecular mobility of solid deuterated tert-butyl alcohol (TBA) has been studied over a broad temperature range (103–283 K) by means of solid-state 2H NMR spectroscopy, including both line shape and anisotropy of spin–lattice relaxation analyses. It has been found that, while the hydroxyl group of the TBA molecule is immobile on the 2H NMR time scale (τC > 10(–5) s), its butyl group is highly mobile. The mobility is represented by the rotation of the methyl [CD3] groups about their 3-fold axes (C3 rotational axis) and the rotation of the entire butyl [(CD3)3-C] fragment about its 3-fold axis (C3′ rotational axis). Numerical simulations of spectra line shapes reveal that the methyl groups and the butyl fragment exhibit three-site jump rotations about their symmetry axes C3 and C3′ in the temperature range of 103–133 K, with the activation energies and preexponential factors E1 = 21 ± 2 kJ/mol, k(01) = (2.6 ± 0.5) × 10(12) s(–1) and E2 = 16 ± 2 kJ/mol, k(02) = (1 ± 0.2) × 10(12) s(–1), respectively. Analysis of the anisotropy of spin–lattice relaxation has demonstrated that the reorientation mechanism of the butyl fragment changes to a free diffusion rotational mechanism above 173 K, while the rotational mechanism of the methyl groups remains the same. The values of the activation barriers for both rotations at T > 173 K have the values, which are similar to those at 103–133 K. This indicates that the interaction potential defining these motions remains unchanged. The obtained data demonstrate that the detailed analysis of both line shape and anisotropy of spin–lattice relaxation represents a powerful tool to follow the evolution of the molecular reorientation mechanisms in organic solids.     

Site discrimination in mixed-alkali glasses studied by cross-polarization NMR

Cation-cation interactions are thought to play a significant role in shaping the nonlinear compositional dependence of ionic conductivity, known as the mixed-alkali effect (MAE) in glassy solid electrolytes. For providing a structural rationale of this effect, the discrimination of various cation sites in mixed-alkali glasses is of interest. In the present study, cross-polarization (CP) experiments have been applied to glasses in the system [(Li(2)O)x(Na(2)O)(1-x)](0.3)[B(2)O(3)]0.7 to discriminate between alkali ions by virtue of different heteronuclear (7)Li-(23)Na dipole-dipole coupling strengths. Cross-polarization studies involving two types of quadrupolar nuclei (both (7)Li and (23)Na have a spin-quantum number I = 3/2) are complicated by spin state mixing under radio frequency irradiation and magic-angle spinning (MAS). Therefore careful validation and optimization protocols are reported for the model compound LiNaSO(4) prior to conducting the measurements on the glassy samples. (23)Na -->( 7)Li CP/MAS NMR spectra have been obtained on glasses containing the Na(+) ions as the dilute species. They reveal that those lithium species interacting particularly strongly with sodium ions have the same average (7)Li chemical shift as the entire lithium population; the symmetrical situation applies to the (23)Na nuclei at the sodium rich end of the composition range. On the other hand, a clear site discrimination is afforded by temperature-dependent static (23)Na -->( 7)Li CP experiments, indicating that the Li ions that are most strongly interacting with sodium ions are strongly immobilized. This finding provides the first direct experimental evidence for the proposed secondary mismatch concept invoked for explaining the strong MAE in the dilute foreign ion limit.     

Molecular complexation and binding studied by electrophoretic NMR spectroscopy

Electrophoretic mobilities obtained on a molecularly selective manner by electrophoretic NMR can be used to provide a quantitative characterization of the composition and stoichiometry of molecular complexes. This is demonstrated in complexes formed by uncharged cyclodextrins which attain an electrophoretic mobility upon inclusion of charged surfactants.     

Polymorphism and dynamics of MBBA as studied by NMR

We report proton NMR experiments on the liquid crystal material N-(p-methoxybenzylidene) p-n-butylaniline (MBBA) at 100MHz in the temperature range 110-350 K. The phase diagram was investigated by means of second moment and spin-lattice relaxation measurements in order to establish connections between dynamics and phase transitions. The results show that in a slow cooling experiment, two processes contribute to the relaxation, a slow ethyl group motion together with reorientation of the methyl groups. For the glassy nematic state, as well as for the phases observed after reheating a quenched sample, only methyl rotation is observed. The correlation times of these various mechanisms were determined and the results compared with those obtained by previous NMR and dielectric analysis.     

Conformation of 2,5-substituted 1,3,2-dioxaborinanes studied by NMR

The conformation of a series of 2,5-substituted 1,3,2-dioxaborinanes is studied by PMR. The preferred conformation for the majority of compounds has an equatorial group on atom C₍₅₎. The 5-nitroderivative exists in the conformation with an axial nitro group.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 8, pp. 1107–1109, August, 1990.