Respiration depends on a lung surfactant layer to facilitate changes in surface area at the air-water interface. The 79-amino acid lung surfactant protein SP-B, normally present in dimer form, is essential to the functioning of lung surfactant. One important aspect of lung surfactant function is the recruitment and cycling of surfactant material to and from bilayer reservoirs. Full utilization of surfactant material available in multilamellar reservoirs presumably requires the formation of contacts between adjacent bilayer surfaces and the formation of connections between leaflets of a given surfactant bilayer. Some fragments of SP-B, including the N-terminal segment SP-B1-25, mimic the effects of full SP-B on model surfactant monolayers and in preparations applied in surfactant-deficient animal model studies. In this work, all-atom molecular dynamics simulations have been carried out on DPPC/POPG model bilayers containing multiple copies of SP-B1-25 initially oriented along the bilayer surface, across the bilayer, or randomly as well as on model bilayers containing SP-B1-9 randomly oriented on the bilayer surfaces. These results show that, over the microsecond durations of the simulations, SP-B1-25 remains largely helical and can be accommodated along the bilayer surface, where it would be available to interact with adjacent bilayers, or across the bilayer which results in local perturbation of the bilayer surface. These observations provide some insight into the possible capacity of the N-terminal segment of SP-B to mediate both interbilayer and intrabilayer interactions that are presumably involved in the lipid assembly reorganization necessary to facilitate surfactant recruitment and recycling from bilayer reservoirs.

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.

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.

The histidine-rich antimicrobial peptides (AMPs) Gad-1 and Gad-2, from paralogous genes in cod, provide an opportunity to examine the effect of charge and non-electrostatic 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 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 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 peptide sequence than with 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 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.

The interaction between the quadrupole moment of a deuteron and the electric field gradient of a carbon-deuterium bond on a lipid acyl chain perturbs the Zeeman splitting of the spin-1 deuteron in an orientation-dependent manner. Partial averaging of that perturbation by asymmetric reorientation of the acyl chains in lipid bilayers provides a powerful tool for probing and characterizing model membrane orientational order and phase behaviour. This chapter begins with a review of the quadrupole interaction as it pertains to H-2 solid-state NMR spectroscopy in lipid bilayers. It then goes on to describe how H-2 solid-state NMR spectroscopy is used to obtain acyl chain orientational order parameter profiles for liquid crystal phase lipid bilayers and to distinguish disordered and ordered lipid bilayer phases. The chapter continues with a discussion of how H-2 solid-state NMR spectroscopy has been used to characterize the phase behaviour of mixed-lipid bilayers and ends with a discussion of important contributions made to the determination of sterol-lipid phase diagrams using this approach.

Gad-1 and Gad-2 are helical, histidine-rich antimicrobial peptides (AMPs) from paralogous genes in cod. N-15 and H-2 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.

The lipid bilayer disrupting effect of antimicrobial peptides (AMPs) has been widely studied in model-lipid systems by applying biophysical techniques such as 2H NMR spectroscopy. Real bacteria cell envelopes contain non-lipid components, such as peptidoglycan, and thus it is important to assess the effects of such non-lipid components on the lipid-disrupting effects of AMPs. To this end, our group and other have developed methods that promote uptake of deuterium-labeled acyl chains in bacterial cells to produce 2H-membrane-enriched Bacillus subtilis. In this work, we studied changes in the static 2H NMR spectra of B. subtilis induced by the AMPs MSI-78 and BP100. Addition of both AMPs resulted in the increase of lipid acyl chain disorder consistent with disruption of the bacterial membrane. In addition, the peptide to lipid molar ratios (P:L) that give rise to observable effects fall between the P:L molar ratios necessary to generate membrane disruption in model-lipid-only systems and the P:L molar ratios needed to inhibit bacterial cell growth. This observation supports a role for the non-lipid components in modulating the AMP-lipid interactions.

Antimicrobial Peptides (AMPs) have been studied for more than two decades because of their promise to help overcome the problem of resistance to conventional antibiotics. However, AMPs have not been as successful as hoped, likely because we lack a detailed understanding of their mechanisms of action. Many biophysical studies of AMPs are performed in model membrane systems, composed of just one or two lipid components. Such studies have shown that membrane permeabilization is a common mechanism of action. Yet we don’t fully understand their connection between the behavior of AMPs in model lipids, where membrane permeabilization is seen and the behavior in real bacteria, where AMPs are observed to inhibit cell growth. Bacteria have an extra non-lipid component in their cell envelopes. It is thus possible that interactions between AMPs and non-lipid components of the cell envelope are important to their mechanisms of action. Specifically, the focus of this study is to find out if the lipopolysaccharide (LPS) outer membrane layer of Gram-negative bacteria promotes or inhibits AMP-induced membrane disruption. This work employed the magainin analog, MSI-78. We disrupt the lipopolysaccharide layer of Gram-negative bacteria (E.coli) via chelation of the stabilizing divalent cations. Then, we use deuterium NMR of deuterated intact bacteria to observe how AMP-induced acyl chain disruption is affected by LPS layer destabilization. In addition to 2H NMR, we do light microscopy and flow cytometry to confirm the EDTA disruption of LPS.

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.

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.

Specific activity against many different pathogens is one of the reasons why antimicrobial peptides (AMPs) are tantalizing candidates to address the current crisis of antibiotic-resistant infections. Nevertheless, many questions about the exact pathogenic mechanisms of AMPs remain unsolved. Biophysical studies on AMPs have shown a destabilizing effect on lipid bilayers. In addition, studies performed in whole bacterial cells have shown that some AMPs may have additional targets different from the lipid bilayer. Thus, it is possible that the destabilizing effect of at least some AMPs on membranes may be a collateral effect of transiting through the membrane on the way to an intra-cellular target, or that there is more than one mechanism by which AMPs harm target cells. Moreover, understanding the role of non-lipid components of the bacterial envelope in the interaction AMPs with bacterial membranes could help us better understand how AMPs selectively target pathogenic bacteria. In this work, we study the role of the peptidoglycan layer in the interaction AMPs with the bacterial membrane. To do so, we implemented 2H NMR in intact Bacillus subtilis cells. In the absence of AMP, the 2H NMR spectrum of the labeled bacterial membrane is a superposition of doublets characteristic of fast axially symmetric chain reorientation. Prominent shoulders, at +/-12 kHz, reflect an order parameter profile having a plateau near the headgroup end of the chain. In the presence of AMP, intensity shifts from the plateau spectral region to smaller quadrupole splittings suggesting a peptide-induced disordering of the bacterial membrane. Finally, we show that for MSI-78 and BP100, peptidoglycan disruption does not affect the AMPs' ability to affect the lipid membrane of intact cells.

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.

Antimicrobial peptides (AMPs) are a group of small peptides with antimicrobial effects against pathogens and have been well studied because of their promise to be part of the solution to the rising problem of antibiotic resistance. Biophysical studies with AMPs in model lipid systems are commonly used to study AMPs' permeabilizing effect on lipid bilayers. However, it is not clear if this membrane-permeabilizing characteristic is the only mechanism of cell killing. Studies suggest that at least some AMPs have additional targets, different from the lipid bilayer. This studies lead to the suggestion that membrane permeabilization is just part of a multi-hit mechanism of AMPs, or even just a collateral effect. Additionally, AMP interactions with non-lipid cell envelope components of bacteria may be important in modifying how well AMPs are able to disrupt the lipid membrane. In order to connect studies of AMPs in model lipid systems to the more complex real bacterial cell envelopes, we have deuterium-labeled the membranes of the gram-positive bacteria Bacillus subtilis and used 2H NMR to study how lipid acyl chain order in its membranes is affected by treatment with AMPs. We have also observed 2H NMR spectra from Bacillus subtilis in which the peptidoglycan layer has been disrupted. This has allowed us to investigate how disruption of the peptidoglycan layer affects bacterial lipid chain order and the AMP/bacteria interaction.

Due to their activity against different pathogens and low toxicity to host cells, antimicrobial peptides (AMPs) are a promising approach to address the bacterial antimicrobial resistance. Biophysical methods have been used to observe the permeabilizing effect of AMPs on model membranes with the same lipid composition as the bacteria cell wall (BCW). In order to fully understand the antimicrobial properties of these peptides, it also necessary to investigate how AMPs interact with other relevant bacterial cell wall components including peptidoglycan and lipopolysaccharide. One way to better understand AMP interactions with non-lipidic components is to observe their effects on the complete bacterial cell wall. 2H Nuclear Magnetic Resonance (2H NMR) can be used to measure the orientational order parameters of lipid acyl chain segments. In this work, we have used 2H NMR to study the effect of AMPs on lipids in the membranes of whole bacteria. We use two techniques to prepare 2H labeled bacteria with isotope-labeled lipids introduced into the bacterial cell wall. These preparation techniques allow us to use 2H NMR to compare the orientational order parameter in the bacteria cell wall lipid bilayer of living wild type cells with and without AMP present. The effects two AMPs, either MSI-78 or CAME (Cepropin A (1-8) and melittin (1-10)), on both gram-negative (Escherichia coli) and gram-positive (Bacillus Subtilis) bacteria have been studied. In the presence of both MSI-78 and CAME, the 2H NMR spectral shape changes in ways that correspond to a decrease in the lipid acyl chain order parameter.

Antimicrobial peptides (AMPs) are an important component of the innate immune system of many different organisms. Their cationic nature and amphipathic structure make them well suited to interact with and perturb bacterial membranes. A major driving force in the binding of AMPs to bacterial membranes is the electrostatic interaction between positively charged residues of AMPs and anionic lipids of the bacterial membrane. Since histidine has a pKa ∼ 6, histidine-rich AMPs can exhibit greater activity at low pH, when the histidine is positively charged, as compared to at neutral pH, when the histidine is neutral. While Gad-1 and Gad-2 are both histidine-rich AMPs, only Gad-2 exhibits pH-dependent activity against E. coli. Hence, it was surprising to find that neither peptide exhibited pH-dependent membrane disruption in model membranes, when probed by 2H NMR. Thus, we hypothesized the differences in activity might be related to differences in binding affinity. In order to probe the binding, we carried out zeta potential measurements with both Gad-1 and Gad-2 at pH 7.0 and pH 5.0. Additionally, 15N solid state NMR measurements were carried out to establish the topology of the Gad peptides in the bilayer. Together these studies can provide a better understanding of membrane-peptide interactions and the effect of pH on these interactions.

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.

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.

Antimicrobial polypeptides (AMPs) are an important component of the innate immune system of many different organisms and are generally amphipathic and cationic in nature. Electrostatic interactions between positively charged residues of the AMP and negatively charged lipids in pathogen membranes are an important component of AMP mechanism and specificity. AMPs such as Gad-1 and Gad-2 that are rich in histidine, an amino acid that is uncharged at neutral pH and positively charged at slightly acidic pH, can thus exhibit pH dependent activity. 2H solid state NMR spectroscopy was used to study the effect of Gad-1 and Gad-2 on model membranes at different pH values. The addition of peptides to the model membrane system POPE/POPG-d31 (3:1) results in a change in the splitting of spectrum both at pH 7 and pH 5. Further investigation of peptide effects on lipid acyl chain motion were determined through calculations of orientational order parameter values (SCD). The order parameter profile demonstrated that Gad-1 causes more lipid acyl chain disordering than Gad-2, at both pH 7 and pH 5. These results are consistent with Gad-1's greater activity in assays for bacterial growth inhibition. On the other hand, while Gad-2 is more active at inhibiting bacterial growth at low pH than at neutral pH, its effect on lipid acyl chain disorder is the same at pH 7 and pH 5.

Maintenance of a functional surfactant layer requires the transfer of material between bilayer reservoirs and the surface-active layer. It is believed that SP-B facilitates the interlayer contact and mixing implicit in such activity. SP-B's role in promoting these contacts was investigated though the use of bilayered micelle mixtures containing short- and long-chain lipids. Upon warming, these bicellar mixtures progressively coalesce into more extended structures and are thus an interesting system in which to study the capacity of polypeptides to promote interactions between lipid structures. 2H NMR was used to identify the capacity of perturbation by an SP-B fragment (SP-B63-78) upon bicellar mixtures of DMPC-d54/DMPG/DHPC and DMPC-d54/DMPC/DHPC in nominal 3:1:1 molar ratios. In the presence of mixtures containing anionic lipids (DMPC-d54/DMPG/DHPC), SP-B63-78 (concentration approximately 10% of lipid weight) was found to lower the temperature at which coalescence to extended lamellar structures occurred. Conversely, when anionic lipids were replaced with zwitterionic lipids (DMPC-d54/DMPC/DHPC), SP-B63-78 did not perturb the temperature at which the transition to extended lamellar structures occurred. These results indicate that the interaction of SP-B63-78 with model membranes is dependent upon the presence of anionic lipids and further suggest a mechanism by which full length SP-B may interact with membranes.