Solid-state Deuterium Nuclear Magnetic Resonance Research Articles

Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications

Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling. We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution 31P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase.

Deuterium solid-state NMR quadrupolar order rotating frame relaxation with applications to amyloid-β fibrils.

We describe a new method for measuring molecular dynamics based on the deuterium solid-state nuclear magnetic resonance (NMR) quadrupolar order rotating frame relaxation rateR1ρ,Q under static conditions. The observed quadrupolar order coherence is created using the broad-band Jeener-Broekaert excitation and is locked with a weak radio frequency (RF) field. We describe the experimental and theoretical approaches and show applications to a selectively deuterated valine side chain of the phosphorylated amyloid-β (1-40) fibrils phosphorylated at the serine-8 position. The R1ρ,Q rate is sensitive to the rotameric exchange mode. For biological samples, the low spin-lock field in the 5- to 10-kHz range has the advantage of avoiding sample heating and dehydration. Thus, it provides an alternative to approaches based on single-quantum coherence, which require larger spin-lock fields.

Phenanthroline Covalent Organic Framework Electrodes for High-Performance Zinc-Ion Supercapattery

Aqueous zinc-ion batteries and capacitors are potentially competitive grid-scale energy storage devices because of their great features such as safety, environmental friendliness, and low cost. Herein, a completely new phenanthroline covalent organic framework (PA-COF) was synthesized and introduced in zinc-ion supercapatteries (ZISs) for the first time. Our as-synthesized PA-COF shows a high capacity of 247 mAh g–1 at a current density of 0.1 A g–1, with only 0.38% capacity decay per cycle during 10 000 cycles at a current density of 1.0 A g–1. Although covalent organic frameworks (COFs) are attracting great attention in many fields, our PA-COF has been synthesized using a new strategy involving the condensation reaction of hexaketocyclohexanone and 2,3,7,8-phenazinetetramine. Detailed mechanistic investigations, through experimental and theoretical methods, reveal that the phenanthroline functional groups in PA-COF are the active zinc ion storage sites. Furthermore, we provide evidence for the cointercalation of Zn2+ (60%) and H+ (40%) into PA-COF using inductively coupled plasma atomic emission spectroscopy and deuterium solid-state nuclear magnetic resonance (NMR). We believe that this study opens a new avenue for COF material design for zinc-ion storage in aqueous ZISs.

Charge and Peptide Concentration as Determinants of the Hydrogel Internal Aqueous Environment.

Self-assembling peptides are a promising class of biomaterials with desirable biocompatibility and versatility. In particular, the oligopeptide (RADA)4, consisting of arginine (R), alanine (A), and aspartic acid (D), self-assembles into nanofibers that develop into a three-dimensional hydrogel of up to 99.5% (w/v) water; yet, the organization of water within the hydrogel matrix is poorly understood. Importantly, peptide concentration and polarity are hypothesized to control the internal water structure. Using variable temperature deuterium solid-state nuclear magnetic resonance (2H NMR) spectroscopy, we measured the amount of bound water in (RADA)4-based hydrogels, quantified as the non-frozen water content. To investigate how peptide polarity affects water structure, five lysine (K) moieties were appended to (RADA)4 to generate (RADA)4K5. Hydrogels at 1 and 5% total peptide concentration were prepared from a 75:25 (w/w) blend of (RADA)4:(RADA)4K5 and similarly analyzed by 2H NMR. Interestingly, at 5% peptide concentration, there was lower mobile water content in the lysinated versus the pristine (RADA)4 hydrogel. Regardless of the presence of lysine, the 5% peptide concentration had higher non-frozen water content at temperatures as low as 217 ± 1.0 K, suggesting that bound water increases with peptide concentration. The bound water, though non-frozen, may be strongly bound to the charged lysine moiety to appear as immobilized water. Further understanding of the factors controlling water structure within hydrogels is important for tuning the transport properties of bioactive solutes in the hydrogel matrix when designing for biomedical applications.

Solid state deuterium NMR study of LKα14 peptide aggregation in biosilica.

In nature, organisms including diatoms, radiolaria, and marine sponges use proteins, long chain polyamines, and other organic molecules to regulate the assembly of complex silica-based structures. Here, the authors investigate structural features of small peptides, designed to mimic the silicifying activities of larger proteins found in natural systems. LKα14 (Ac-LKKLLKLLKKLLKL-C), an amphiphilic lysine/leucine repeat peptide with an α-helical secondary structure at polar/apolar interfaces, coprecipitates with silica to form nanospheres. Previous 13C magic angle spinning studies suggest that the tetrameric peptide bundles that LKα14 is known to form in solution may persist in the silica-complexed form, and may also function as catalysts and templates for silica formation. To further investigate LKα14 aggregation in silica, deuterium solid-state nuclear magnetic resonance (2H ssNMR) was used to establish how leucine side-chain dynamics differ in solid LKα14 peptides isolated from aqueous solution, from phosphate-buffered solution, and in the silica-precipitated states. Modeling the 2H ssNMR line shapes probed the mechanisms of peptide preaggregation and silica coprecipitation. The resulting NMR data indicates that the peptide bundles in silica preserve the hydrophobic interior that they display in the hydrated solid state. However, NMR data also indicate free motion of the leucine residues in silica, a condition that may result from structural deformation of the aggregates arising from interactions between the surface lysine side chains and the surrounding silica matrix.

Uncovering the Rotation and Translational Mobility of Benzene Confined in UiO-66 (Zr) Metal–Organic Framework by the 2H NMR–QENS Experimental Toolbox

A combination of experimental quasi-elastic neutron scattering (QENS) and deuterium solid-state nuclear magnetic resonance (2H NMR) techniques was used to uncover the molecular mobility of benzene confined in UiO-66 (Zr) MOF with a 3D cage-window-cage type porous network topology. We have shown that tetrahedral and octahedral cages of UiO-66 offer notably different states of confinement for benzene. Below 163 K, the guest molecules of benzene are represented by two different dynamic states: in the smaller (tetrahedral) cage the benzene is able to exhibit only anisotropic C6 rotation and some limited librations. In the octahedral cage, there is enough space for additional C2 axial rotation and the isotropic random reorientation. Rotational motions have been characterized by the rate constants and corresponding activation energies. The two dynamic states merge as the temperature increases due to translational jump diffusion with the limiting step being the passage through the window between the cages. Both ...

Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2H solid-state NMR

Nuclear magnetic resonance (NMR) is commonly used to probe the effect of antimicrobial agents on bacterial membranes using model membrane systems. Ideally, considering the complexity of membranes, the interaction of molecules with membranes should be studied in vivo. The interactions of two antimicrobial peptides (AMPs) with intact Escherichia coli and Bacillus subtilis were investigated using deuterium solid-state NMR. Specifically, we studied caerin 1.1 and aurein 1.2 isolated from the skin of Australian tree frogs. The minimal inhibitory concentration value for E. coli and B. subtilis was about 100μg/mL and 30μg/mL, respectively, for both peptides. A protocol to deuterate the membrane phospholipids of non-mutated B. subtilis was established using deuterated palmitic acid. 2H NMR spectra combined with spectral moment analysis support the interaction of the two AMPs with the hydrophobic core of the bacterial membranes. The presence of peptides decreased the order of the lipid acyl chains for both E. coli and B. subtilis, but at higher peptide concentrations for the Gram(+) bacteria. This may be explained by the presence of other cell wall components, such as the negatively-charged teichoic and lipoteichoic acids in the peptidoglycan, which would interact with the AMPs and decrease their actual concentration on the membrane surface. The mechanism of action of the AMPs thus depends on their local concentration as well as the membrane environment. The differences between the AMPs interaction with E. coli and B. subtilis reveal the importance of studying intact bacteria.

Imidazolium Trifluoromethanesulfonate sPEEK Composites for Anhydrous High Temperature Proton Exchange Membrane Fuel Cells

Proton exchange membranes fuel cells (PEMFCs) are excellent energy carriers for numerous applications such as automobiles. Current standard operating conditions (<80°C) of PEMFCs are unfortunately optimized in a way that leads to catalyst poisoning by carbon monoxide. Higher operating temperatures (>120°C) are desirable, and can be achieved with alternative materials that conduct protons anhydrously via a Grotthuss mechanism. Alternative materials for an electrolyte would be ionic salts such as solid acids. Solid acids are chemically, thermally and electrochemically stable and conduct protons under anhydrous conditions. 1-3 Previous solid acids studied contained phosphate functional groups where it was shown that these systems have good conductivity in anhydrous conditions.4 Such phosphates possess substantial proton conductivity; however high temperatures can promote an irreversible condensation into pyrophosphates, which lowers conductivity. In this study sulfonate-based solid acids are used. Heterocycles such as imidazole are paired with acids such as trifluoromethanesulfonic acid to produce acidic salts, e.g. imidazolium trifluoromethane sulfonate (IFMS). These conductive salts cannot be placed into a fuel cell since they do not have the capability to form thin films therefore, a polymer support must be used. Previous investigations focused on non-ionic polymers, such as Teflon,, where decent proton conductivity was obtained.5 The polymer support chosen here is sulfonated polyetheretherketone (sPEEK), a well-known proton-conducting polymer, which offers a host architecture for cooperative proton transport. By itself sPEEK requires a high degree of sulfonation to be ionically conductive; however it is known to swell excessively in water at high degrees of sulfonation.6 Preliminary proton conductivity measurements at 120°C and 0% RH show 4.4x10-4 to 5.9x10-3 S/cm with degrees of salt loading between 32-50 percent, as determined by thermal gravimetric analysis. Initial ion exchange capacity (IEC) studies showed that sPEEK itself possesses 2.1±0.2 mmol/g of available sulfonate groups with the loading of 32% IFMS the IEC increased to 3.8±0.2 mmol/g. With the addition of the salt, there is a proportional increase the number of available sulfonate groups that can contribute to proton conductivity. Solid state nuclear magnetic resonance (NMR) methods can investigate dynamics on a localized scale to probe mobility of proton sites. Methods include 1H magic angle spinning (MAS) NMR and 2H static NMR studies, which reveal details of timescale and geometry of exchange processes. Deuterium solid state NMR is a good probe of dynamic processes in the solid state, as the characteristic static lineshapes are highly sensitive to the dynamic processes such as molecular reorientations, and can be accurately modeled to extract rates of chemical exchange.2 These measurements will compare dynamics and conductivity in the salts themselves, sPEEK and the salt-sPEEK composites. Figure 1 depicts IFMS at 300 K and 350 K. When the temperature is increased the line shape narrows and additional features are observed. These features correspond to an increased jump rate from the ring flip within the salt. Jump rates can be fitted to extract activation energies of the ring flip. References (1) Noda, A.; Susan, A. B.; Kudo, K.; Mitsushima, S.; Hayamizu, K.; Watanabe, M. Journal of Physical Chemistry B 2003, 107, 4024. (2) Sekhon, S. S.; Park, J. S.; Cho, E.; Yoon, Y. G.; Kim, C. S.; Lee, W. Y. MACROMOLECULES 2009, 42, 2054. (3) Watanabe, M.; Tsurumi, K.; Mizukami, T.; Nakamura, T.; Stonehart, P. Journal of The Electrochemical Society 1994, 141, 2659. (4) Traer, J. W. Journal of Physical Chemistry B 2007, 111, 5602. (5) Yan, Z. B.; De Almeida, N. E.; Traer, J. W.; Goward, G. R. Phys. Chem. Chem. Phys. 2013, 15, 17983. (6) Huang, R. Y. M.; Shao, P.; Burns, C. M.; Feng, X. Journal of Applied Polymer Science 2001, 82, 2651.

Hydrogen storage in Pd nanocrystals covered with a metal-organic framework.

Hydrogen is an essential component in many industrial processes. As a result of the recent increase in the development of shale gas, steam reforming of shale gas has received considerable attention as a major source of H2, and the more efficient use of hydrogen is strongly demanded. Palladium is well known as a hydrogen-storage metal and an effective catalyst for reactions related to hydrogen in a variety of industrial processes. Here, we present remarkably enhanced capacity and speed of hydrogen storage in Pd nanocrystals covered with the metal-organic framework (MOF) HKUST-1 (copper(II) 1,3,5-benzenetricarboxylate). The Pd nanocrystals covered with the MOF have twice the storage capacity of the bare Pd nanocrystals. The significantly enhanced hydrogen storage capacity was confirmed by hydrogen pressure-composition isotherms and solid-state deuterium nuclear magnetic resonance measurements. The speed of hydrogen absorption in the Pd nanocrystals is also enhanced by the MOF coating.

Interaction between the Marine Sponge Cyclic Peptide Theonellamide A and Sterols in Lipid Bilayers As Viewed by Surface Plasmon Resonance and Solid-State 2H Nuclear Magnetic Resonance

Theonellamides (TNMs) are members of a distinctive family of antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. Recently, it has been shown that TNMs recognize 3β-hydroxysterol-containing membranes, induce glucan overproduction, and damage cellular membranes. However, to date, the detailed mode of sterol binding at a molecular level has not been determined. In this study, to gain insight into the mechanism of sterol recognition of TNM in lipid bilayers, surface plasmon resonance (SPR) experiments and solid-state deuterium nuclear magnetic resonance ((2)H NMR) measurements were performed on theonellamide A (TNM-A). SPR results revealed that the incorporation of 10 mol % cholesterol or ergosterol into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes significantly enhances the affinity of the peptide for the membrane, particularly in the initial binding to the membrane surface. These findings, together with the fact that binding of TNM-A to epicholesterol (3α-cholesterol)-containing liposomes and pure POPC liposomes was comparably weak, confirmed the preference of the peptide for the 3β-hydroxysterol-containing membranes. To further establish the formation of the complex of TNM-A with 3β-hydroxysterols in lipid bilayers, solid-state (2)H NMR measurements were conducted using deuterium-labeled cholesterol, ergosterol, or epicholesterol. The (2)H NMR spectra showed that TNM-A significantly inhibits the fast rotational motion of cholesterol and ergosterol, but not epicholesterol, therefore verifying the direct complexation between TNM-A and 3β-hydroxysterols in lipid bilayers. This study demonstrates that TNM-A directly recognizes the 3β-OH moiety of sterols, which greatly facilitates its binding to bilayer membranes.

Effect of Lipid Bilayer Composition on the Membrane-Binding Ability and Depth of Insertion of Antimicrobial Piscidin 1: A Solid-State Deuterium NMR Study

Piscidins constitute a family of amphipathic, cationic, antimicrobial peptides (AMPs) found in the skin, gills and gastrointestinal tracts of hybrid striped bass. Unstructured in water, they become alpha-helical in the presence of phospholipids. It is believed that their ability to disrupt phospholipid bilayers is related to their antimicrobial effects on yeasts and bacteria. Interestingly, the minimum inhibitory concentrations of piscidin tend to be higher for yeasts than most bacteria. Here, we used Circular Dichroism (CD) and deuterium solid-state Nuclear Magnetic Resonance (ssNMR) to investigate the membrane-binding ability and depth of insertion of piscidin 1 in different membrane mimetic systems. Aligned lipid bilayer preparations were made using 3:1 POPC/POPG-d31 (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol-d31) and 3:1 POPC/POPS-d31 (1-palmitoyl 2-oleoyl phosphatidylserine-d31) to mimic bacterial and yeast cell membranes, respectively. To test the effects of ergosterol, a sterol found in yeasts, we added 20% ergosterol (molar ratio) to the lipid mixtures. We hypothesized that both PS and ergosterol would reduce the ability of piscidin to bind and disrupt bilayers.Deuterium quadrupolar splittings, which were obtained for deuterons in the methylene and terminal methyl groups of the phospholipid acyl chains, were used to assess the disordering effect of piscidin on the acyl chains. Our results suggest that the type of anionic lipid component (e.g., PG versus PS) does not affect acyl chain disordering by piscidin while ergosterol appears to decrease its disordering effect. The data also give evidence that membrane-bound piscidin is located close to the phospholipid headgroups. Overall, these studies help us gain some insight into specific physicochemical features of lipid membranes that affect piscidin-lipid interactions. The implications in terms of piscidin's mode of action will be discussed.

Solid State 2H NMR Analysis of Furanose Ring Dynamics in DNA Containing Uracil

DNA damage has been implicated in numerous human diseases, particularly cancer, and the aging process. Single-base lesions, such as uracil, in DNA can be cytotoxic or mutagenic and are recognized by a DNA glycosylase during the process of base excision repair. Increased dynamic properties in lesion-containing DNAs have been suggested to assist recognition and specificity. Deuterium solid-state nuclear magnetic resonance (SSNMR) has been used to directly observe local dynamics of the furanose ring within a uracil:adenine (U:A) base pair and compared to a normal thymine:adenine (T:A) base pair. Quadrupole echo lineshapes, <T(1Z)>, and <T(2e)> relaxation data were collected, and computer modeling was performed. The results indicate that the relaxation times are identical within the experimental error, the solid lineshapes are essentially indistinguishable above the noise level, and our lineshapes are best fit with a model that does not have significant local motions. Therefore, U:A base pair furanose rings appear to have essentially identical dynamic properties as a normal T:A base pair, and the local dynamics of the furanose ring are unlikely to be the sole arbiter for uracil recognition and specificity in U:A base pairs.

Orientation and Motion of Tryptophan Interfacial Anchors in Membrane-Spanning Peptides

The tryptophans of integral membrane proteins have been suggested to play specific roles as "interfacial anchors", based on their preference for a location near the lipid head groups. Still, the underlying mechanism behind this behavior remains unclear. NMR experiments can provide an important tool to study this interaction in an actual bilayer environment. Here solid-state deuterium nuclear magnetic resonance was used to study the tryptophans in membrane-spanning model peptides from the WALP family (acetyl-GWW(LA)nWWA-ethanolamide with n = 5 and 6.5) in samples of mechanically aligned dimyristoylphosphatidylcholine (DMPC) bilayers. The data indicate that the tryptophans near the C-terminal end of the peptide display a significantly different behavior from those near the N-terminus. This is reflected prominently in a large difference in the motion experienced by the indoles at either end of the peptide, highlighting the directionality of the helix. Nevertheless, our observations indicate high levels of motional freedom for all tryptophans in these membrane spanning domains that exceed the dynamics for the helix itself. These observations signify that steric and dynamic features of the polypeptide context modulate the tryptophan anchoring in the membrane interface. Measurements of WALP19 in the ether-linked DMPC analogue ditetradecylphosphatidylcholine (missing the lipid carbonyls) show very similar Trp dynamics and suggest similar orientations for some or all of the tryptophans. This suggests that the lipid acyl chain carbonyls play at most a minor role in the anchoring interaction between these Trp residues and the DMPC interfacial region.

Template induced chiral ordering in nematic liquid crystalline materials: A deuterium nuclear magnetic resonance study

Glancing angle deposition is used to create porous liquid crystal alignment layers with high aspect-ratio, three-dimensional features. We use solid-state deuterium nuclear magnetic resonance to determine the alignment of alpha deuterated 4′-pentyl-4-cyanobiphenyl in a 3.2μm thick SiO2 film comprised of independent, helical structures. Comparing measured spectra with models, we determine that the liquid crystalline molecules exhibit chiral ordering dictated by the features of the template films. In the plane of deposition the liquid crystalline molecules tilt away from the substrate normal at an average angle of 51°, which is commensurate with the helical features of the chiral template.

Effect of Rotational Mobility on Photoelectron Transfer: Comparison of Two Zeolite Topologies

Nuclear magnetic resonance (NMR) spectroscopy and time-resolved diffuse reflectance (TRDR) have been combined to study the effects of constrained rotational motion on the rates of photoinduced electron-transfer reactions within zeolites. By synthesizing tris(2,2‘-bipyridine) ruthenium (II) [Ru(bpy)32+] within the large cages of zeolites Y and EMT, it was possible to directly investigate the effect of zeolite cage size on molecular motion of Ru(bpy)32+ and the influence of the cage size on the rate of intrazeolitic electron transfer. Deuterium solid-state NMR shows that zeolite Y imposes restraint on molecular rotation of Ru(bpy)32+, while zeolite EMT allows for motion. Temperature-dependent studies show the ability to freeze or induce motion of Ru(bpy)32+ within zeolite EMT while motion within zeolite Y remains unaffected, even at elevated temperatures. For zeolite EMT, an increase in the rate of the photoinduced forward and back electron transfer from Ru(bpy)32+ to methyl viologen was noted and correlated with access, rotational motion, and favorable orbital overlap. The overall photochemical charge separation efficiencies for the intrazeolitic Ru(bpy)32+-bipyridinium reactions were similar for both zeolites.

Chain segment order in ultrathin polymer films: A deuterium NMR study

The orientational order of monomerically thin films of polydimethylsiloxane melt deposited on the surfaces of cylindrical pores is investigated in this paper by solid-state deuterium nuclear magnetic resonance. Spectra demonstrate substantial orientation of the methyl groups by the surface. The quadrupolar splitting of the oriented film component saturates below one monomer layer, but decreases stepwise for thicker films. Comparisons of experimental and simulated spectra suggest a flat chain conformation in the first polymer monolayer.

Solid-state deuterium nuclear magnetic resonance of the methyl dynamics of poly(α-methylstyrene) and polymethylphenylsilane

The methyl-d3 dynamics of two relatively similar polymers, poly(α-methylstyrene) (PAMS-d3) and polymethylphenylsilane (PMPS-d3), are investigated via deuterium NMR relaxation experiments. Our analysis of the relaxation data uses the entire solid-echo spectra to maximize the precision of the experiments with regard to the information available on the methyl dynamics. The analysis is novel in that it does not use M∞ or M0 to fit the relaxation data. Additionally, the three-site symmetric jump model is shown to not have an observable azimuthal angular dependence for T1 relaxation. The methyl dynamics are quantified with τm, σ, and f which are the log-average correlation time, half-height full-width (base 10) of a log-normal distribution of reorientation rates, and the anisotropy of the relaxation, respectively. The anisotropy parameter, f, is based on a serial combination of the rotational diffusion and symmetric three-site jump reorientation of a methyl deuteron. This serial model coupled with a distribution of τc’s has a minimal number of parameters that have physical meaning and quantify the observations of our relaxation data. Generally, at similar temperatures the methyl reorientation in PAMS-d3 is at least 100 times slower than that of PMPS-d3. For both polymers, both τm and σ decrease with increasing temperature, resulting in activation energies of 12 and 5 kJ/mol for PAMS-d3 and PMPS-d3, respectively. Also, with increasing temperature a mechanistic change from three-site jump to rotational diffusion is observed and quantified. This information, along with that of other studies, suggests that the PAMS-d3 methyls have highly restrictive environments that may be closely coupled to phenyl-ring reorientation.

Chain mobility in the amorphous region of nylon 6 observed under active uniaxial deformation

A specially designed solid-state deuterium nuclear magnetic resonance probe was used to examine the effect of uniaxial elongation on the chain mobility in the amorphous region of semicrystalline nylon 6. In measurements conducted near the glass transition temperature, there was measurable deformation-induced enhancement of the mobility of the amorphous chains up to the yield point. This enhanced mobility decayed once deformation was stopped. Enhanced mobility was not observed in deformation at room temperature. The mechanics of deformation can be explained by the Robertson model for glassy polymers near the glass transition temperature, which states that applied stress induces liquid-like behavior in the polymer chains.

Membrane binding and pore formation of the antibacterial peptide PGLa: thermodynamic and mechanistic aspects.

The antibacterial peptide PGLa exerts its activity by permeabilizing bacterial membranes whereas eukaryotic membranes are not affected. To provide insight into the selectivity and the permeabilization mechanism, the binding of PGLa to neutral and negatively charged model membranes was studied with high-sensitivity isothermal titration calorimetry (ITC), circular dichroism (CD), and solid-state deuterium nuclear magnetic resonance ((2)H NMR). The binding of PGLa to negatively charged phosphatidylcholine (PC)/phosphatidylglycerol (PG) (3:1) vesicles was by a factor of approximately 50 larger than that to neutral PC vesicles. The negatively charged membrane accumulates the cationic peptide at the lipid-water interface, thus facilitating the binding to the membrane. However, if bulk concentrations are replaced by surface concentrations, very similar binding constants are obtained for neutral and charged membranes (K approximately 800-1500 M(-)(1)). Membrane selectivity is thus caused almost exclusively by electrostatic attraction to the membrane surface and not by hydrophobic insertion. Membrane insertion is driven by an exothermic enthalpy (DeltaH approximately -11 to -15 kcal/mol) but opposed by entropy. An important contribution to the binding process is the membrane-induced random coil --> alpha-helix transition of PGLa. The peptide is random coil in solution but adopts an approximately 80% alpha-helical conformation when bound to the membrane. Helix formation is an exothermic process, contributing approximately 70% to the binding enthalpy and approximately 30% to the free energy of binding. The (2)H NMR measurements with selectively deuterated lipids revealed small structural changes in the lipid headgroups and in the hydrocarbon interior upon peptide binding which were continuous over the whole concentration range. In contrast, isothermal titration calorimetry of PGLa solutions with PC/PG(3:1) vesicles gave rise to two processes: (i) an exothermic binding of PGLa to the membrane followed by (ii) a slower endothermic process. The latter is only detected at peptide-to-lipid ratios >17 mmol/mol and is paralleled by the induction of membrane leakiness. Dye efflux measurements are consistent with the critical limit derived from ITC measurements. The endothermic process is assigned to peptide pore formation and/or lipid perturbation. The enthalpy of pore formation is 9.7 kcal/mol of peptide. If the same excess enthalpy is assigned to the lipid phase, the lipid perturbation enthalpy is 180 cal/mol of lipid. The functional synergism between PGLa and magainin 2 amide could also be followed by ITC and dye release experiments and is traced back to an enhanced pore formation activity of a peptide mixture.

A deuterium NMR study of guest molecular dynamics in tris(5-acetyl-3-thienyl) methane inclusion compounds

Deuterium nuclear magnetic resonance (NMR) spectra and spin-lattice relaxation times (T1) are used to investigate the guest molecular dynamics of tris(5-acetyl-3-thienyl) methane (TATM) inclusion compounds. The seven guests: acetonitrile, nitromethane, dimethyl sulfoxide, benzene, mesitylene, ortho-xylene, and para-xylene show a wide variety of motional behaviour. The reorientation of acetonitrile in TATM and nitromethane in TATM were both modelled as precession on a cone, the base of which is more elliptical in shape for nitromethane, as would be expected considering their molecular symmetries. DMSO in TATM does not undergo any reorientation other than methyl rotation at the temperatures investigated. At low temperatures, the 2H lineshape and the deuterium spin-lattice relaxation time both depend on the rate of methyl rotation. Activation barriers of 11.7 (±0.4) kJ/mol and 11.2 (±0.5) kJ/mol were found from the two techniques, respectively. Benzene undergoes sixfold reorientation about the principal molecular axis; however, the rate is still greater than 108 Hz down to 113 K. The spin-lattice relaxation time profile does not reach a minimum on decreasing temperature to 112 K, while the slope of this plot provides an activation energy of 4.1 (±0.4) kJ/mol for the sixfold reorientation. The deuterium NMR spectra of mesitylene-d9 in TATM can be simulated using a model where the guest occupies two different sites in the TATM lattice, with the guest molecules performing in-plane C3 rotation either very rapidly (k &gt; 108 Hz) or very slowly (k &lt; 103 Hz), with the relative populations of each changing with temperature. Another model proposed suggests the possibility that there is a continuous distribution of motional rates, the median of which is increasing with temperature. This second model is more realistic; however, too many parameters are present to consider a detailed fit. Finally, both o-xylene and p-xylene are rigidly held in the TATM clathrate, while rotation of the methyl groups is rapid. Key words: inclusion compounds, solid state deuterium NMR spectroscopy, molecular dynamics.