Role of lipopolysaccharide in antimicrobial and cell penetrating peptide membrane interactions probed by deuterium NMR of whole cells.

Understanding how non-lipid components of bacteria affect antimicrobial peptide (AMP)-induced membrane disruption is important for a comprehensive understanding of AMP mechanisms and informing AMP-based drug development. This study investigates how lipopolysaccharide (LPS) affects membrane disruption by the AMP MSI-78 and compares the results to the effect of TP2, a cell-penetrating peptide that crosses membrane bilayers without permeabilizing them. We destabilize the LPS layer of Escherichia coli (E. coli) cells via chelation of the stabilizing divalent cations. 2H NMR spectra of E. coli demonstrate that EDTA concentrations of 2.5 mM and 9.0 mM alone have very minor effects on lipid acyl chain order. Interestingly, we find that E. coli pre-treated with 9.0 mM EDTA before treatment with MSI-78 are more sensitive to AMP-induced acyl chain disruption, indicating that intact LPS reduces MSI-78-induced membrane disruption in E. coli. Surprisingly, we also found that at the level of 2H NMR, the peptide-induced acyl chain disruption is similar for MSI-78 and TP2, although MSI-78 permeabilizes the bilayer and TP2 does not. Furthermore, LPS disruption appears to protect the bacteria from TP2, although it sensitizes them to MSI-78.

Role of lipopolysaccharide in antimicrobial and cell penetrating peptide membrane interactions probed by deuterium NMR of whole cells.

Understanding how non-lipid components of bacteria affect antimicrobial peptide (AMP)-induced membrane disruption is important for a comprehensive understanding of AMP mechanisms and informing AMP-based drug development. This study investigates how lipopolysaccharide (LPS) affects membrane disruption by the AMP MSI-78 and compares the results to the effect of TP2, a cell-penetrating peptide that crosses membrane bilayers without permeabilizing them. We destabilize the LPS layer of Escherichia coli (E. coli) cells via chelation of the stabilizing divalent cations. 2H NMR spectra of E. coli demonstrate that EDTA concentrations of 2.5 mM and 9.0 mM alone have very minor effects on lipid acyl chain order. Interestingly, we find that E. coli pre-treated with 9.0 mM EDTA before treatment with MSI-78 are more sensitive to AMP-induced acyl chain disruption, indicating that intact LPS reduces MSI-78-induced membrane disruption in E. coli. Surprisingly, we also found that at the level of 2H_NMR, the peptide-induced acyl chain disruption is similar for MSI-78 and TP2, although MSI-78 permeabilizes the bilayer and TP2 does not. Furthermore, LPS disruption appears to protect the bacteria from TP2, although it sensitizes them to MSI-78.

2H NMR Study of Polymer Segmental Dynamics at Varying Cross-Linking in Poly(N-isopropylacrylamide) Microgels.

A microscopic understanding of the internal structure and dynamics of poly(N-isopropylacrylamide) (PNIPAM) chains, in microgel colloids, is developed using deuterium NMR (2H NMR) to study deuterated PNIPAM suspensions as functions of temperature and pressure for four cross-linker molar fractions (f). The PNIPAM polymers were labeled with deuterons at the backbone (d3-PNIPAM) or on side chains (d7-PNIPAM). 2H NMR spectra of the d3-PNIPAM suspensions for all cross-linker molar fractions indicated freely moving chains at low temperature and a nearly immobilized fraction above ∼35 °C. Polymer segments in the collapsed phase of the d3-PNIPAM suspension were more mobile than those in the dry powder. This is direct microscopic evidence that the polymer remains significantly hydrated in the collapsed phase, consistent with strong, indirect evidence from recent light scattering and rheology measurements from our laboratory. However, the observation of a small fraction of immobilized segments in the swollen phase for higher cross-linker molar fraction suggests that, particularly for high levels of cross-linking, some polymer is nonhydrated even in the swollen phase. Finally, variable-pressure NMR (up to 90 MPa) showed a slight increase in transition temperature with pressure for lower cross-linker molar fractions and a larger increase in transition temperature with pressure for higher cross-linker molar fractions. This is consistent with a previously reported dependence of collapse transition enthalpy on cross-linker molar fraction.

Role of Charge in Lipid Vesicle Binding and Vesicle Surface Saturation by Gaduscidin-1 and Gaduscidin-2.

The histidine-rich antimicrobial peptides Gad-1 and Gad-2, from paralogous genes in cod, provide an opportunity to examine the effect of charge and nonelectrostatic factors on peptide-vesicle interaction and on peptide antimicrobial activity. In this study, the dependence of vesicle ζ-potential on peptide concentration has been used to examine the binding of these peptides to model vesicle surfaces at pH = 5.0, for which the charges of Gad-1 and Gad-2 are +8 and +5, respectively, and at pH = 7.0, where their charges are +3 and +1, respectively. Interpreting the observed ζ-potential behaviors as examples of Langmuir adsorption isotherms, it is possible to infer the equilibrium constant for peptide-vesicle binding, the fraction of the peptide bound at low peptide concentration, and the maximum peptide-to-lipid ratio when the vesicle surface is saturated at high peptide concentration. For both peptides, higher peptide charge is found to be correlated with a lower fraction of the peptide being bound to vesicle surfaces at low peptide concentration and with a smaller maximum bound peptide-to-lipid ratio at high peptide concentration. The equilibrium binding constant, on the other hand, is more strongly correlated with the peptide sequence than with the charge. Gad-1, which has been shown to be more biologically active than Gad-2, displayed a significantly higher equilibrium binding constant. These observations suggest that while the maximum peptide density on the vesicle surface is limited by electrostatic interactions, the free energy of peptide binding, like the observed antimicrobial activities of the Gad peptides, is also sensitive to other peptide factors which might, for example, influence hydrophobic interactions.

Roles of histidine charge and cardiolipin in membrane disruption by antimicrobial peptides Gaduscidin-1 and Gaduscidin-2.

Gad-1 and Gad-2 are helical, histidine-rich antimicrobial peptides (AMPs) from paralogous genes in cod. 15N and 2H solid state nuclear magnetic resonance (NMR) were used to characterize their lipid-bound structures and lipid interactions. Gad-1 was found to position in-plane in POPC: POPG bilayers. Gad-1 displayed greater effects than Gad-2 on lipid acyl chain order of POPE: POPG and POPE: POPG: CL bilayers, in keeping with its greater activity against E. coli. The effect of Gad-1 and Gad-2 on lipid bilayer order was only weakly affected by changes in pH, and hence changes in histidine charge. This was somewhat surprising for Gad-2 as this peptide's biological activity has been shown to be greater at low pH and thus the finding may point to the existence of functional interactions with non-lipid components of bacteria. The incorporation of cardiolipin into POPE: POPG bilayers in such a way as to preserve the overall charge of the bilayers did not alter Gad-1's effects on lipid acyl chain order parameters, which report on motions on the 10-5 s timescale. When cardiolipin and Gad-1 were both present, there were subtle changes on membrane dynamics at other timescales.

Structural parameters of soft PNIPAM microgel particles as a function of crosslink density.

HYPOTHESIS: The temperature dependences of hydrodynamic radii in thermo-sensitive microgel suspensions, known as collapse curves, are commonly fitted to the benchmark Flory-Rehner theory but parameters obtained often yield little physical insights. Our study of poly(N-isopropylacrylamide) (PNIPAM) microgel suspensions in water is driven by the hypothesis that fitting to Flory-Rehner theory can yield meaningful parameters that separate into ones that are insensitive to crosslink density or deuteration and ones that are not. EXPERIMENTS: Dynamic light scattering (DLS) and rheology experiments were done on 8 microgel variants, protonated and deuterated PNIPAM for four crosslink densities, synthesized under otherwise identical conditions. FINDINGS: Remarkably, polymer volume fractions in the microgel particle at collapse, ϕcollapse, obtained via rheology, are independent of crosslink density. Along with collapse curves from DLS, this determines the temperature dependence of microgel water and polymer volume fractions. Fitting collapse curves to Flory-Rehner theory yields reference polymer volume fractions, ϕ0, associated with microgel particle elasticity. ϕ0 is much lower than ϕcollapse, and increases with crosslink density. For all microgel sample variants, a crossover temperature, where the elastic contribution to osmotic pressure changes sign, is found to approximate the final temperature after microgel synthesis and also to the free polymer θ temperature.

Effects of Amphipathic Polypeptides on Membrane Organization Inferred from Studies Using Bicellar Lipid Mixtures.

SP-B63-78, a lung surfactant protein fragment, and magainin 2, an antimicrobial peptide, are amphipathic peptides with the same overall charge but different biological functions. Deuterium nuclear magnetic resonance has been used to compare the interactions of these peptides with dispersions of 1,2-dimyristoyl- sn-glycero-3-phophocholine (DMPC)/1,2-dihexanoyl- sn-glycero-3-phophocholine (DHPC) (4:1) and DMPC/1,2-dimyristoyl- sn-glycero-3-phopho-(1'-rac-glycerol) (DMPG)/DHPC (3:1:1), two mixtures of long-chain and short-chain lipids that display bicellar behavior. This study exploited the sensitivity of a bicellar system structural organization to factors that modify partitioning of their lipid components between different environments. In small bicelle particles formed at low temperatures, short-chain components preferentially occupy curved rim environments around bilayer disks of the long-chain components. Changes in chain order and lipid mixing, on heating, can drive transitions to more extended assemblies including a magnetically orientable phase at intermediate temperature. In this work, neither peptide had a substantial effect on the behavior of the zwitterionic DMPC/DHPC mixture. For bicellar mixtures containing the anionic lipid DMPG, the peptide SP-B63-78 lowered the temperature at which magnetically orientable particles coalesced into more extended lamellar structures. SP-B63-78 did not promote partitioning of the zwitterionic and anionic long-chain lipid components into different environments. Magainin 2, on the other hand, was found to promote separation of the anionic lipid, DMPG, and the zwitterionic lipid, DMPC, into different environments for temperatures above 34 °C. The contrast between the effects of these two peptides on the lipid mixtures studied appears to be consistent with their functional roles in biological systems.

Divide & Conquer: Surfactant Protein SP-C and Cholesterol Modulate Phase Segregation in Lung Surfactant.

Lung surfactant (LS) is an essential system supporting the respiratory function. Cholesterol can be deleterious for LS function, a condition that is reversed by the presence of the lipopeptide SP-C. In this work, the structure of LS-mimicking membranes has been analyzed under the combined effect of SP-C and cholesterol by deuterium NMR and phosphorus NMR and by electron spin resonance. Our results show that SP-C induces phase segregation at 37°C, resulting in an ordered phase with spectral features resembling an interdigitated state enriched in dipalmitoylphosphatidylcholine, a liquid-crystalline bilayer phase, and an extremely mobile phase consistent with small vesicles or micelles. In the presence of cholesterol, POPC and POPG motion seem to be more hindered by SP-C than dipalmitoylphosphatidylcholine. The use of deuterated cholesterol did not show signs of specific interactions that could be attributed to SP-C or to the other hydrophobic surfactant protein SP-B. Palmitoylation of SP-C had an indirect effect on the extent of protein-lipid perturbations by stabilizing SP-C structure, and seemed to be important to maximize differences among the lipids participating in each phase. These results shed some light on how SP-C-induced lipid perturbations can alter membrane structure to sustain LS functionality at the air-liquid interface.

Protocols for Studying the Interaction of MSI-78 with the Membranes of Whole Gram-Positive and Gram-Negative Bacteria by NMR.

Antimicrobial peptides (AMPs) may interact with a variety of target cell components, including the lipid bilayer, non-lipidic cell envelope components, and/or intracellular targets. However, most biophysical experiments aimed at elucidating the detailed mechanism of AMPs are limited to simple model membrane systems and neglect potentially functional interactions between AMPs and non-lipidic cell components. One of the biophysical techniques commonly used to study how AMPs interact with lipid bilayers is solid-state deuterium NMR. In this chapter we provide protocols to prepare deuterium-labeled intact Gram-negative and Gram-positive bacteria and to observe these samples using solid-state deuterium NMR. Such experiments have the potential to provide important information about how non-lipidic cell envelope components modulate AMP interactions with the cytoplasmic membrane of bacteria.

Effect of an Anionic Lipid on the Barotropic Behavior of a Ternary Bicellar Mixture.

Dispersions of lipid mixtures comprising long- and short-chain phospholipids (bicellar mixtures) can form small isotropically reorienting particles (bilayered micelles), magnetically orientable stuctures, or unorientable lamellar structures. Application of hydrostatic pressure can also induce interdigitation of the long-chain lipid components. In this work, variable-pressure 2H NMR was used to study the effect of head group charge on the barotropic behavior of bicellar mixtures. Observations at pressures up to 152 MPa and temperatures up to 64 °C were combined with earlier observations at lower pressure and lower temperature to obtain a pressure-temperature phase diagram for DMPC-d54/DMPG/DHPC (3:1:1). In this phase diagram, a region corresponding to small, isotropically reorienting particles at lower temperature and higher pressure is separated from a region corresponding to unorientable lamellar organization, at higher temperature and lower pressure, by a band in which the magnetically orientable phase is stable below ∼100 MPa and in which an interdigitated gel phase is stable above ∼120 MPa. From ∼46 to ∼52 °C, the dispersion transforms directly from the unorientable lamellar to isotropically reorienting particle phases upon isothermal pressurization. The extent to which this behavior reflects the presence of anionic lipid in the long-chain fraction of this mixture is illustrated by comparison with spectral series obtained during isothermal pressurization of DMPC-d54/DHPC (4:1) and DMPC-d54/DMPG/DHPC (2.7:1.3:1) at selected temperatures. These observations show how electrostatic interactions at a bilayer surface can affect the balance between hydrophobic and hydrophilic interactions that is reflected by a dispersion's barotropic phase behavior.

Response to hydrostatic pressure of bicellar dispersions containing an anionic lipid: pressure-induced interdigitation.

Bicellar dispersions of chain perdeuterated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC-d54), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), with molar ratios of 3:1:1, were studied using variable-pressure (2)H NMR spectroscopy at hydrostatic pressures up to 125 MPa. Upon warming of the dispersions, spectra at ambient pressure indicated a progressive coalescence from small bilayered disks undergoing isotropic reorientation to more extended micellar structures in which spectra indicated anisotropic reorientation and, under some conditions, magnetic orientation and finally to randomly oriented lamellae or multilamellar vesicles. Temperatures for the onsets of anisotropic reorientation and random lamellar orientation increased with pressure at rates of 0.22 and 0.15 °C/MPa, respectively. In the 3.5-T magnetic field used for this work, magnetic orientation within the intermediate phase was not observed at 83 MPa or higher pressures. Comparison of spectra obtained at fixed pressure showed significant asymmetry between behaviors upon warming and cooling. For samples of DMPC-d54/DMPG/DHPC (3:1:1), but not DMPC-d54/DHPC (4:1), a persistent interdigitated phase was formed after repeated cooling from high temperature at 83 MPa. This is likely a metastable phase and might reflect kinetic trapping of the short-chain lipid component, DHPC, in a nonequilibrium spatial distribution as temperature is lowered at high pressure. Bicellar dispersions typically behave differently upon warming and cooling, and these observations could provide some insight into the observed behaviors in such systems. This work also suggests the possibility of trapping bicellar dispersions in persistent nonequilibrium morphologies.

Role of the N-terminal seven residues of surfactant protein B (SP-B).

Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B) is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the "insertion sequence". These studies employed a construct of SP-B, SP-B (1-25,63-78), also called Super Mini-B, which is a 41-residue peptide with internal disulfide bonds comprising the N-terminal 7-residue insertion sequence and the N- and C-terminal helices of SP-B. Circular dichroism, solution NMR, and solid state (2)H NMR were used to study the structure of SP-B (1-25,63-78) and its interactions with phospholipid bilayers. Comparison of results for SP-B (8-25,63-78) and SP-B (1-25,63-78) demonstrates that the presence of the 7-residue insertion sequence induces substantial disorder near the centre of the lipid bilayer, but without a major disruption of the overall mechanical orientation of the bilayers. This observation suggests the insertion sequence is unlikely to penetrate deeply into the bilayer. The 7-residue insertion sequence substantially increases the solution NMR linewidths, most likely due to an increase in global dynamics.

Interaction of the C-terminal peptide of pulmonary surfactant protein B (SP-B) with a bicellar lipid mixture containing anionic lipid.

The hydrophobic lung surfactant SP-B is essential for respiration. SP-B promotes spreading and adsorption of surfactant at the alveolar air-water interface and may facilitate connections between the surface layer and underlying lamellar reservoirs of surfactant material. SP-B63-78 is a cationic and amphipathic helical peptide containing the C-terminal helix of SP-B. (2)H NMR has been used to examine the effect of SP-B63-78 on the phase behavior and dynamics of bicellar lipid dispersions containing the longer chain phospholipids DMPC-d 54 and DMPG and the shorter chain lipid DHPC mixed with a 3∶1∶1 molar ratio. Below the gel-to-liquid crystal phase transition temperature of the longer chain components, bicellar mixtures form small, rapidly reorienting disk-like particles with shorter chain lipid components predominantly found around the highly curved particle edges. With increasing temperature, the particles coalesce into larger magnetically-oriented structures and then into more extended lamellar phases. The susceptibility of bicellar particles to coalescence and large scale reorganization makes them an interesting platform in which to study peptide-induced interactions between lipid assemblies. SP-B63-78 is found to lower the temperature at which the orientable phase transforms to the more extended lamellar phase. The peptide also changes the spectrum of motions contributing to quadrupole echo decay in the lamellar phase. The way in which the peptide alters interactions between bilayered micelle structures may provide some insight into some aspects of the role of full-length SP-B in maintaining a functional surfactant layer in lungs.

Dependence of bicellar system phase behavior and dynamics on anionic lipid concentration.

Bicellar dispersions of chain perdeuterated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC-d54) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) were prepared with the molar fraction of DHPC held fixed at 20% and varying amounts of DMPC replaced by the anionic lipid 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG). (2)H NMR spectra were examined to assess the effect of added DMPG on mixture phase behavior and morphology. Quadrupole echo decay and quadrupole-Carr-Purcell-Mieboom-Gill echo train measurements provided information about slow motions contributing to echo decay in the high temperature phases. The spectra and quadrupole echo decay properties of DMPC-d54/DHPC (4:1) and DMPC-d54/DMPG/DHPC (3:1:1) were qualitatively similar. With increasing DMPG concentration, the transition between the magnetically orientable phase and the higher temperature phase became increasingly distinct, and the spectral shape and echo decay characteristics of the high temperature bicellar phase became increasingly similar to those of DMPC-d54 in the liquid crystalline phase. The observation that DMPG changes spectra in the orientable phase incrementally while increasing the distinction between the orientable and high temperature bicellar phases provides new insights into how DMPG influences bicellar mixture morphology.

²H solid-state nuclear magnetic resonance investigation of whole Escherichia coli interacting with antimicrobial peptide MSI-78.

A key aspect of the activity of antimicrobial peptides (AMPs) is their interaction with membranes. Efforts to elucidate their detailed mechanisms have focused on applying biophysical methods, including nuclear magnetic resonance (NMR), to AMPs in model lipid systems. However, these highly simplified systems fail to capture many of the features of the much more complex cell envelopes with which AMPs interact in vivo. To address this issue, we have designed a procedure to incorporate high levels of (2)H NMR labels specifically into the cell membrane of Escherichia coli and used this approach to study the interactions between the AMP MSI-78 and the membranes of intact bacteria. The (2)H NMR spectra of these membrane-deuterated bacteria can be reproduced in the absence and presence of MSI-78. Because the (2)H NMR data provide a quantitative measure of lipid disorder, they directly report on the lipid bilayer disruption central to the function of AMPs, in the context of intact bacteria. Addition of MSI-78 to the bacteria leads to decreases in the order of the lipid acyl chains. The molar peptide:lipid ratios required to observe the effects of MSI-78 on acyl chain order are approximately 30 times greater than the ratios needed to observe effects in model lipid systems and approximately 100 times less than the ratios required to observe inhibition of cell growth in biological assays. The observations thus suggest that MSI-78 disrupts the bilayer even at sublethal AMP levels and that a large fraction of the peptide does not actually reach the inner membrane.

Dynamic properties of bicellar lipid mixtures observed by rheometry and quadrupole echo decay.

In bicellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), the transition from isotropic reorientation to partial orientational order, on warming, is known to coincide with a sharp increase in viscosity. In this work, cone-and-plate rheometry, (2)H NMR spectroscopy, and quadrupole echo decay observations have been used to obtain new insights into the dynamics of phases observed in bicellar DMPC/DHPC mixtures. Samples with 25% of the DMPC component deuterated were used to correlate rheological measurements with phase behavior observed by (2)H NMR spectroscopy. Mixtures containing only normal DMPC (DMPC/DHPC) or only chain perdeuterated DMPC (DMPC-d(54)/DHPC) were used to refine rheology and quadrupole echo decay measurements respectively. The viscosity peaked at 4-9 Pa·s, just above the isotropic-to-nematic transition, and then dropped as samples were warmed through the nematic-to-lamellar transition. Quadrupole echo decay times above the nematic-to-lamellar transition were significantly longer than typically observed in the liquid crystalline phase of saturated lipid multilamellar vesicles. This may indicate a damping of slow bilayer undulations resulting from the coupling of opposite bilayer surfaces by DHPC-lined pores.

Orientation and dynamics of synthetic transbilayer polypeptides containing GpATM dimerization motifs.

Deuterium NMR spectroscopy was used to study how the positioning of a dimerization motif within a transbilayer polypeptide influences its orientation and dynamics in bilayers. Three polypeptide variants comprising glycophorin A transmembrane (GpATM) dimerization motifs incorporated into lysine-terminated poly-leucine-alanine helices were mixed into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine multilamellar vesicles. The variants differed in orientation of the motif segment around the helix axis with respect to the peptide ends. Polypeptides were labeled with methyl-deuterated alanines at positions that were identically situated relative to the peptide ends (Ala-20 and Ala-22) and at two positions within the motif. An analysis of quadrupole splittings revealed similar tilts and orientations of the peptide ends for all three variants, suggesting that average orientations were dominated by interactions at the bilayer surface. For one variant, however, fast orientational fluctuations about the helix axis were significantly smaller. This may indicate some perturbation of peptide dynamics and conformation by interactions that are sensitive to the motif orientation relative to the peptide ends. For the variant that displayed distinct dynamics, one orientation consistent with observed splittings corresponded to the motif being situated such that its two glycines were particularly accessible to adjacent peptides.

Modifications to surfactant protein B structure and lipid interactions under respiratory distress conditions: consequences of tryptophan oxidation.

These studies detail the altered structure-function relationships caused by oxidation of surfactant protein B (SP-B), a mode of damage thought to be important in acute respiratory distress syndrome (ARDS), a common and frequently fatal condition. An 18-residue fragment comprising the N-terminal helix of SP-B was investigated in oxidized and unmodified forms by solution and solid-state nuclear magnetic resonance (NMR), circular dichroism (CD), and molecular dynamics (MD) simulation. Taken together, the results indicate that tryptophan oxidation causes substantial disruptions in helical structure and lipid interactions. The structural modifications induced by tryptophan oxidation were severe, with a reduction in helical extent from approximately three helical turns to, at most, one turn, and were observed in a variety of solvent environments, including sodium dodecyl sulfate (SDS) micelles, dodecyl phosphocholine (DPC) micelles, and a 40% hexafluoro-2-propanol (HFIP) aqueous solution. The unmodified peptide takes on an orientation within lipid bilayers that is tilted approximately 30° away from an in-plane position. Tryptophan oxidation causes significant modifications to the peptide-lipid interactions, and the peptide likely shifts to a more in-plane orientation within the lipids. Interestingly, the character of the disruptions to peptide-lipid interactions caused by tryptophan oxidation was highly dependent on the charge of the lipid headgroup.

Bicellar mixture phase behavior examined by variable-pressure deuterium NMR and ambient pressure DSC.

Variable-pressure deuterium nuclear magnetic resonance ((2)H NMR) has been used to study the pressure-temperature phase diagram of bicellar mixtures containing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). Spectra were obtained for DMPC-d(54)/DHPC (3:1), DMPC-d(54)/DHPC (4.4:1), DMPC/DHPC-d(22) (3:1), and DMPC/DHPC-d(22) (4.4:1) in the range 10-68 degrees C at ambient pressure, 66 MPa, 102 MPa, and 135 MPa. Isotropic-to-nematic and nematic-to-lamellar transition temperatures were found to rise with pressure at approximately 0.15 and approximately 0.14 degrees C/MPa, respectively, for DMPC-d(54)/DHPC (3:1) and at at approximately 0.19 and approximately 0.18 degrees C/MPa, respectively, for DMPC-d(54)/DHPC (4.4:1). Pressure had little effect on the range of DMPC-d(54) chain orientational order through the nematic phase temperature range, but the behavior of chain orientational order at the nematic-to-lamellar transition was found to vary slightly with pressure. Comparison of differential scanning calorimetry (DSC) observations with ambient-pressure (2)H NMR observations of DMPC-d(54) in the bicellar mixtures suggests that absorption of heat persists for a few degrees above the onset of axially symmetric DMPC-d(54) reorientation.

The effect of a C-terminal peptide of surfactant protein B (SP-B) on oriented lipid bilayers, characterized by solid-state 2H- and 31P-NMR.

SP-B(CTERM), a cationic, helical peptide based on the essential lung surfactant protein B (SP-B), retains a significant fraction of the function of the full-length protein. Solid-state (2)H- and (31)P-NMR were used to examine the effects of SP-B(CTERM) on mechanically oriented lipid bilayer samples. SP-B(CTERM) modified the multilayer structure of bilayers composed of POPC, POPG, POPC/POPG, or bovine lipid extract surfactant (BLES), even at relatively low peptide concentrations. The (31)P spectra of BLES, which contains approximately 1% SP-B, and POPC/POPG with 1% SP-B(CTERM), look very similar, supporting a similarity in lipid interactions of SP-B(CTERM) and its parent protein, full-length SP-B. In the model systems, although the peptide interacted with both the oriented and unoriented fractions of the lipids, it interacted differently with the two fractions, as demonstrated by differences in lipid headgroup structure induced by the peptide. On the other hand, although SP-B(CTERM) induced similar disruptions in overall bilayer orientation in BLES, there was no evidence of lipid headgroup conformational changes in either the oriented or the unoriented fractions of the BLES samples. Notably, in the model lipid systems the peptide did not induce the formation of small, rapidly tumbling lipid structures, such as micelles, or of hexagonal phases, the observation of which would have provided support for functional mechanisms involving peptide-induced lipid flip-flop or stabilization of curved lipid structures, respectively.