Tiago Mendes Ferreira

Segmental order parameters in a nonionic surfactant lamellar phase studied with 1H–13C solid-state NMR

A lyotropic nonionic lamellar system composed of pentaethyleneglycol mono n-dodecyl ether and D(2)O was studied using natural abundance (13)C NMR under magic-angle spinning. Applying a two-dimensional recoupling method proposed by Dvinskikh (R-PDLF), (1)H-(13)C dipolar couplings were estimated over a range of temperatures (300-335 K), thus enabling analysis of structural changes in the liquid crystalline system. The results obtained are used to correlate the conformation and mobility of local sites in the surfactant molecule with overall changes in the lamellar structure.

Model-free estimation of the effective correlation time for C-H bond reorientation in amphiphilic bilayers: (1)H-(13)C solid-state NMR and MD simulations.

Molecular dynamics (MD) simulations give atomically detailed information on structure and dynamics in amphiphilic bilayer systems on timescales up to about 1 μs. The reorientational dynamics of the C-H bonds is conventionally verified by measurements of (13)C or (2)H nuclear magnetic resonance (NMR) longitudinal relaxation rates R1, which are more sensitive to motional processes with correlation times close to the inverse Larmor frequency, typically around 1-10 ns on standard NMR instrumentation, and are thus less sensitive to the 10-1000 ns timescale motion that can be observed in the MD simulations. We propose an experimental procedure for atomically resolved model-free estimation of the C-H bond effective reorientational correlation time τe, which includes contributions from the entire range of all-atom MD timescales and that can be calculated directly from the MD trajectories. The approach is based on measurements of (13)C R1 and R1ρ relaxation rates, as well as (1)H-(13)C dipolar couplings, and is applicable to anisotropic liquid crystalline lipid or surfactant systems using a conventional solid-state NMR spectrometer and samples with natural isotopic composition. The procedure is demonstrated on a fully hydrated lamellar phase of 1-palmitoyl-2-oleoyl-phosphatidylcholine, yielding values of τe from 0.1 ns for the methyl groups in the choline moiety and at the end of the acyl chains to 3 ns for the g1 methylene group of the glycerol backbone. MD simulations performed with a widely used united-atom force-field reproduce the τe-profile of the major part of the acyl chains but underestimate the dynamics of the glycerol backbone and adjacent molecular segments. The measurement of experimental τe-profiles can be used to study subtle effects on C-H bond reorientational motions in anisotropic liquid crystals, as well as to validate the C-H bond reorientation dynamics predicted in MD simulations of amphiphilic bilayers such as lipid membranes.

Molecular conformation and bilayer pores in a nonionic surfactant lamellar phase studied with (1)h-(13)c solid-state NMR and molecular dynamics simulations.

The structure of the lamellar phase of aqueous pentaethylene glycol mono-n-dodecyl ether (C12E5) surfactant at various temperatures and molar fractions is studied by using united atom molecular dynamics simulations and nuclear magnetic resonance measurements. Namely, the simulation model is used to interpret the magnitude and temperature dependence of experimental C-H order parameter profiles in terms of the molecular conformation and orientation. Our simulations suggest that the low order parameters that are generally measured in poly(ethylene oxide) surfactant bilayers are due to the presence of bilayer pores throughout the entire lamellar phase region.

NMR Studies of Nonionic Surfactants

NMR has contributed greatly to our current understanding of self-association, phase behaviour, and molecular dynamics in aqueous systems of oligo(ethylene oxide) mono-alkyl ether (CmEn) nonionic surfactants, which are extensively used in both basic scientific studies and technical applications as emulsifying agents and detergents. This review provides a comprehensive but concise overview of the various NMR techniques that have been applied to nonionic surfactants. We describe conventional experimental procedures, such as the measurement of quadrupole splittings, nuclear relaxation times, and self-diffusion coefficients to characterize liquid crystalline phases, micelles, and microemulsions, as well as more advanced imaging and diffusion-diffusion 2D correlation approaches to investigate the structure of phase-separated systems and the spatial organization of anisotropic liquid crystalline domains on the micro- to millimetre length scales.

Acyl Chain Disorder and Azelaoyl Orientation in Lipid Membranes Containing Oxidized Lipids

Oxidized phospholipids occur naturally in conditions of oxidative stress and have been suggested to play an important role in a number of pathological conditions due to their effects on a lipid membrane acyl chain orientation, ordering, and permeability. Here we investigate the effect of the oxidized phospholipid 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) on a model membrane of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using a combination of (13)C-(1)H dipolar-recoupling nuclear magnetic resonance (NMR) experiments and united-atom molecular dynamics (MD) simulations. The obtained experimental order parameter SCH profiles show that the presence of 30 mol % PazePC in the bilayer significantly increases the gauche content of the POPC acyl chains, therefore decreasing the thickness of the bilayer, although with no stable bilayer pore formation. The MD simulations reproduce the disordering effect and indicate that the orientation of the azelaoyl chain is highly dependent on its protonation state with acyl chain reversal for fully deprotonated states and a parallel orientation along the interfacial plane for fully protonated states, deprotonated and protonated azelaoyl chains having negative and positive SCH profiles, respectively. Only fully or nearly fully protonated azelaoyl chain are observed in the (13)C-(1)H dipolar-recoupling NMR experiments. The experiments show positive SCH values for the azelaoyl segments confirming for the first time that oxidized chains with polar termini adopt a parallel orientation to the bilayer plane as predicted in MD simulations.

Applications of Solid-State NMR Spectroscopy for the Study of Lipid Membranes with Polyphilic Guest (Macro)Molecules

The incorporation of polymers or smaller complex molecules into lipid membranes allows for property modifications or the introduction of new functional elements. The corresponding molecular-scale details, such as changes in dynamics or features of potential supramolecular structures, can be studied by a variety of solid-state NMR techniques. Here, we review various approaches to characterizing the structure and dynamics of the guest molecules as well as the lipid phase structure and dynamics by different high-resolution magic-angle spinning proton and 13C NMR experiments as well as static 31P NMR experiments. Special emphasis is placed upon the incorporation of novel synthetic polyphilic molecules such as shape-persistent T- and X-shaped molecules as well as di- and tri-block copolymers. Most of the systems studied feature dynamic heterogeneities, for instance those arising from the coexistence of different phases; possibilities for a quantitative assessment are of particular concern.

Effect of cholesterol on the molecular structure and transitions in a clinical-grade lung surfactant extract

Significance Cholesterol is currently removed from most lung surfactant extract preparations used in clinical applications. Cholesterol-depleted samples are also used in most in vitro studies of the lung surfactant layer. In our study we have performed a detailed molecular characterization of the structure of the lung surfactant with regard to cholesterol content and temperature. We show that cholesterol has a strong impact on the phase behavior, structure, and dynamics of the lung surfactant system even at low concentrations. We emphasize the importance of controlling the cholesterol content of the lung surfactant system studied to better mimic the endogenous lung surfactant and develop better preparations for clinical treatments. The lipid–protein film covering the interface of the lung alveolar in mammals is vital for proper lung function and its deficiency is related to a range of diseases. Here we present a molecular-level characterization of a clinical-grade porcine lung surfactant extract using a multitechnique approach consisting of H1–C13 solid-state nuclear magnetic spectroscopy, small- and wide-angle X-ray scattering, and mass spectrometry. The detailed characterization presented for reconstituted membranes of a lung extract demonstrates that the molecular structure of lung surfactant strongly depends on the concentration of cholesterol. If cholesterol makes up about 11% of the total dry weight of lung surfactant, the surfactant extract adopts a single liquid-ordered lamellar phase, Lα(o), at physiological temperatures. This Lα(o) phase gradually changes into a liquid-disordered lamellar phase, Lα(d), when the temperature is increased by a few degrees. In the absence of cholesterol the system segregates into one lamellar gel phase and one Lα(d) phase. Remarkably, it was possible to measure a large set of order parameter magnitudes |SCH| from the liquid-disordered and -ordered lamellar phases and assign them to specific C–H bonds of the phospholipids in the biological extract with no use of isotopic labeling. These findings with molecular details on lung surfactant mixtures together with the presented NMR methodology may guide further development of pulmonary surfactant pharmaceuticals that better mimic the physiological self-assembly compositions for treatment of pathological states such as respiratory distress syndrome.

Cholesterol-like effects of a fluorotelomer alcohol incorporated in phospholipid membranes

Fluorocarbon amphiphiles are anthropogenic substances widely used in diverse applications such as food packaging, clothing or cookware. Due to their widespread use and non-biodegradability, these chemicals are now ubiquitous in the natural world with high propensity to bioaccumulate in biological membranes, wherein they may affect microscopic properties. Here, we test the hypothesis that a typical fluorocarbon amphiphile can affect lipid membranes similarly to cholesterol by investigating the effect of 1H,1H,2H,2H-perfluoro-1-decanol (8:2 FTOH) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membranes. Using solid-state nuclear magnetic resonance spectroscopy, differential scanning calorimetry and confocal microscopy, we present a consistent set of independent experimental evidences supporting this hypothesis, namely that upon incorporation of 8:2 FTOH, (i) a condensing effect on the acyl chains occurs in the fluid phase, (ii) coexistence of two membrane phases is observed below melting, and (iii) the melting temperature of DPPC varies no more than approximately ±1 °C up to a concentration of 40 mol% of 8:2 FTOH. The condensing effect is quantified by means of advanced dipolar recoupling solid-state NMR experiments and is found to be of approximately half the magnitude of the cholesterol effect at the same concentration.