There is a considerable interest in understanding the function of antimicrobial peptides (AMPs), but the details of their mode of action is not fully understood. This motivates extensive efforts in determining structural and mechanistic parameters for AMP’s interaction with lipid membranes. In this study we show that oriented-sample 31P solid-state NMR spectroscopy can be used to probe the membrane perturbations and -disruption by AMPs. For two AMPs, alamethicin and novicidin, we observe that the majority of the lipids remain in a planar bilayer conformation but that a number of lipids are involved in the peptide anchoring. These lipids display reduced dynamics. Our study supports previous studies showing that alamethicin adopts a transmembrane arrangement without significant disturbance of the surrounding lipids, while novicidin forms toroidal pores at high concentrations leading to more extensive membrane disturbance.

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Recently, ether lipids have been introduced as long-term stable alternatives to the more natural, albeit easier degradable, ester lipids in the preparation of oriented lipid bilayers and bicelles for oriented-sample solid-state NMR spectroscopy. Here we report that ether lipids such as the frequently used 14-O-PC (1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine) may induce significant changes in the structure and dynamics, including altered interaction between peptides and lipids relative to what is observed with the more conventionally used DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers. Such effects are demonstrated for the antimicrobial peptide novicidin, for which 2D separate-local-field NMR and circular dichroism experiments reveal significant structural/conformational differences for the peptide in the two different lipid systems. Likewise, we observe altered secondary structure and different temperature-dependent membrane anchoring for the antimicrobial peptide alamethicin depending on whether the peptide is reconstituted into ester or ether lipids. Such observations are not particularly surprising considering the significant difference of the lipids in the phosphorus headgroup and they may provide important new insight into the delicate peptide-membrane interactions in the systems studied. In contrast, these observations reinforce the need to carefully consider potential structural changes in addition to long-term stability prior to the selection of membrane environment of membrane proteins in the analysis of their structure and dynamics. In more general terms, the results underscore the necessity in structural biology to address both the protein and its environments in studies relating structure to function.

We present a method to calculate (31)P solid-state NMR spectra based on the dynamic input from extended molecular dynamics (MD) simulations. The dynamic information confered by MD simulations is much more comprehensive than the information provided by traditional NMR dynamics models based on, for example, order parameters. Therefore, valuable insight into the dynamics of biomolecules may be achieved by the present method. We have applied this method to study the dynamics of lipid bilayers containing the antimicrobial peptide alamethicin, and we show that the calculated (31)P spectra obtained with input from MD simulations are in agreement with experiments under a large range of different sample conditions, including vesicles and oriented samples with and without peptides. We find that the changes in the (31)P spectra upon addition of peptide stem from lipids with reduced diffusion due to peptide-lipid interactions.

Mixed phospholipid micelles (bicelles) are widely applied in nuclear magnetic resonance (NMR) studies of membrane proteins in solution, as they can solubilize these proteins and provide a membrane-like environment. In this work, the structure of bicelles of dihexanoyl phosphatidyl choline (DHPC) and dimyristoyl phosphatidyl choline (DMPC) at different ratios was determined by small-angle X-ray scattering (SAXS) at 37 °C. Samples with concentrations as applied for NMR measurements with 28 wt % lipids were diluted to avoid concentration effects in the SAXS data. The DMPC/DHPC ratio within the bicelles was kept constant by diluting with solutions of finite DHPC concentrations, where the concentration of free DHPC is the same as in the original solution. Absolute-scale modeling of the SAXS data using molecular and concentration constraints reveals a relatively complex set of morphologies of the lipid aggregates as a function of the molar ratio Q of DMPC to DHPC. At Q = 0 (pure DHPC lipids), oblate core–shell micelles are present. At Q = 0.5, the bicelles have a tablet-shaped core–shell cylindrical form with an ellipsoidal cross section. For Q = 1, 2, 3.2, and 4, the bicelles have a rectangular cuboidal structure with a core and a shell, for which the overall length and width increase with Q. At Q = ∞ (pure DMPC), there is coexistence between multilamellar structures and free bilayers. For Q = 1–4, the hydrocarbon core is relatively narrow and the headgroup thickness on the flat areas is larger than that of, respectively, pure DHPC and DMPC, suggesting some mixing of DHPC into these areas and staggering of the molecules. This is further supported by comparisons of the ratio of the areas of rim and flat parts and estimates of the composition of the flat areas.