Lipids were purchased and used without further purification, 1,2-di-oleoylphosphatidylcholine (DOPC) from Avanti Polar Lipids; cholesterol from BDH chemicals Ltd Poole England; and, 98% Ergosterol (nitrogen fixed) from Acros Organics. Amphotericin B was purchased from MP Biomedicals and used without further purification. Natamax was kindly donated by DuPont and lactose was removed via multiple washes in distilled water to yield pure natamycin.

DOPC was mixed with either cholesterol or ergosterol in 7:3 ratios in chloroform/methanol and re-suspended in a working buffer 10 mM HEPES pH 7.0, 10 mM NaCl, 1 mM EDTA (the same buffer was used for all samples). Natamycin was dissolved in methanol and added to the DOPC/sterol lipid film (final ratio of DOPC/sterol/drug was 7:3:1.5), and after solvent removal also re-suspended in the working buffer. Amphotericin B was dissolved in di-methylformamide (DMF) and added to DOPC/Cholesterol lipid film (final ratio 7:3:1.5) and DMF was removed by repeated 7 × dilution with distilled water and centrifugation at 20,000 rpm at 20°C (Beckman coulter Avanti J25) for 30 min. Residual DMF was removed under high vacuum and the sample was re-suspended in the working buffer. All samples were then freeze-thawed a minimum of 5 times each, pelleted at 16,060 g for 15 min (Biofuge pico, Heraeus) and the pellets were loaded in 4 mm MAS NMR rotors.

All solid-state NMR experiments were performed on a Varian 400 MHz (VNMRS) spectrometer using a 4 mm T3 MAS NMR probe (Varian, Palo Alto CA, USA). Cross-polarization, CP, (Hartmann and Hahn, 1962) with proton ramp (Metz et al., 1994) was used to prepare observable ¹³C magnetization. A 96 kHz proton excitation pulse was followed by 3.5 ms of 45 kHz Hartmann-Hahn contact. MAS NMR spectra were acquired using 60 kHz SPINAL decoupling (Fung et al., 2000) to suppress heteronuclear ¹H−¹³C dipolar interactions or using 96 kHz FSLG decoupling (Bielecki et al., 1989). Acquisitions were repeated each 3.5 s with 150 ms acquisition times under SPINAL and 75 ms duration under FSLG. To obtain heteronuclear-decoupled high-resolution spectra, 1024 transients were averaged, while 8192 transients were added when homonuclear decoupling was used. Sample temperature was regulated using balanced heated/vortex tube-cooled gas flow (Ciesielski et al., 2009) and MAS/RF-induced temperature elevation of 4°C was taken into consideration by indirect reference to lipid main transition suppression in DMPC and DPPC liposomes. Sample rotation was set at 5 kHz, which is sufficient to produce well-averaged high-resolution spectra in mobile hydrated lipid systems. Spectral prediction and processing was done using ADC Labs (Toronto, Canada).

High-resolution ¹³C MAS NMR spectra, acquired under SPINAL decoupling from DOPC liposomes containing 30% sterol at 20°C yielded well-resolved resonances. Cholesterol and ergosterol structures are shown in Figure 1 alongside atom numbering used in resonance assignments (Zorin et al., 2010). Pure DOPC membranes were characterized for reference. Samples made with cholesterol produced well-resolved lipid and sterol resonances (Figure 2), which were assigned following (Forbes et al., 1988; Zorin et al., 2010). DOPC/ergosterol samples produced well-resolved lipid peaks but broad and low-intensity ergosterol resonances in most cases (Figure 3), due to intermediate mobility of this sterol in DOPC membranes, which interferes with cross-polarization and homonuclear dipolar decoupling. Resolved ergosterol peaks were assigned according to $Soubias et al. (Soubias and Gawrisch, 2005) and used to monitor the membrane response to antifungals. The same set of samples was studied using ¹³C MAS NMR spectroscopy with FSLG decoupling during signal acquisition, which removes proton degeneracy and provides J-resolved ¹³C spectra for sufficiently mobile molecular segments. Well-resolved couplings were measured for DOPC resonances only, due to the poor resolution of the sterol multiplets.

Molecular interactions in membranes can be followed using ¹³C MAS NMR by following changes in segmental dynamics. Such changes impact longitudinal relaxation, which leads to loss of intensity in resonances from directly interacting groups (Sanghera et al., 2011) or intensity enhancement under CP-MAS where fast molecular mobility interfering with CP or decoupling is moderated in a complex and NMR signal is enhanced. Figure 2 shows ¹³C MAS NMR spectra from DOPC/cholesterol membranes alone (bottom) and in the presence of amphotericin B (middle) or natamycin (top). Amphotericin has a profound effect on membrane dynamics and all lipid and cholesterol resonances appear broadened, indicating that amphotericin B does insert in the membrane and that insertion has a disordering effect leading to increased librational freedom for both DOPC and cholesterol. The linewidth is likely related to motional interference with the proton decoupling, as the lipid CO resonances, which have no directly bonded protons appear less affects. Despite this line broadening, the relative intensity of cholesterol resonances compared to DOPC does not appear significantly altered, which indicates no preferential interaction of amphotericin B with cholesterol over DOPC.

While fast lipid dynamics is dominated by cooperative axial rotation, changes in segmental mobility in response to antifungals can still be observed by ¹³C solid state NMR and segmental dynamics in sterols is even more informative. In the presence of amphotericin B cholesterol core residues c1–6, c9, c11, c14, c15–17 are significantly attenuated, indicating reduced core mobility (Figure 2 middle). Curiously, c12 is attenuated but still observed and methyls c18, c19, and c21, which line the same region of the cholesterol molecule do not appear affected. Amphotericin has little effect on chain residues c20–c25, as well. However, resonance intensity from chain terminal methyl groups c26 and c27 are enhanced, which points to participation of the chain end in the interaction between amphotericin and cholesterol. Chain terminal coupling has been reported between ergosterol terminal methyl groups e27, e28 and amphotericin B in their membrane complex (Umegawa et al., 2012). Together, the data put forward an interaction model, in which the cholesterol ring core plays the leading role in interactions with amphotericin B, chiefly on the c4, c6, c15 ridge and with engagement of the chain end c26, c27.

The impact of natamycin on the spectrum from DOPC/cholesterol membrane is quite different (Figure 2 top). Overall, resonances have retained their width offering good spectral resolution. The impact of natamycin on cholesterol mobility indicates the formation of a molecular complex, which bears resemblance to that between amphotericin B and cholesterol showing ring core and chain-terminal interactions with natamycin. However, distinct differences in segmental dynamics reveal mobility restrictions on all ring methyl groups, as well as on ring A (c1–5, c10) and the base of the chain. This points to natamycin engagement of the cholesterol molecule on the opposite side to that with amphotericin B and involving cholesterol ring 1 (c1–c5) and the chain end (c25–c27), however, also with contact on methyls c18, c18 and not directly with the other rings.

Ergosterol-containing membranes undergo motions near the NMR timescale, which leads to some interference with proton decoupling and cross-polarization and erodes some ergosterol and antibiotic resonances from the ¹³C MAS NMR spectra (Figure 3, bottom). Resolved resonances inform on the impact of antibiotic presence on segmental dynamics through changes in resonance intensity (dependent on the longitudinal relaxation time T₁) by comparison to the repeat delay.

The presence of amphotericin B in the DOPC/ergosterol membrane is associated with attenuation of ergosterol resonances, which is consistent with altered molecular dynamics during complex formation between ergosterol and amphotericin B. This attenuation is most noticeable and specific for resonances e1, e9, e12, and e19, which complement the interfacial ridge of rings A, B, and C in ergosterol along with methyl e19. The opposite side of the molecule, e15, is also attenualted, which appears to link to mobility restrictions in the chain at e20, e22, e24, and e25 (Figure 3, middle). The terminal methyls e27 and e28 are not attenuated but are shifted. This points to an interaction picture, which involves the e1-e9-e12 edge with e19 and the beginning of the chain e20, e22, e24 along with ring D at e15 (Figure 1).

Addition of natamycin to ergosterol/DOPC membranes yields a better resolved spectrum, in which a number of natamycin residues are also observed (Figure 3, top). The attenuation of ergosterol signals in this spectrum is far more widespread involving the same ergosterol ridge e1, e10, e9, e11, as well methyl e19 but also resonances e3 and e4 on ring A, e7 on the opposite side of ring B, e13 and e15 on ring D. Interestingly, akin to amphotericin, natamycin does not attenuate methyl e18. The ergosterol chain is also more profoundly affected with resonances e23, e24, and e25 attenuated, as well as terminal methyls e27 and e28. Curiously, resonance e26 is significantly enhanced, showing that the end of the ergosterol chain undergoes non-cooperative motions near the intermediate timescale that are affected profoundly on natamycin binding. In contrast to amphotericin B, antamycin appears to engage methyl e21 at the beginning of the chain (Figure 1). The impact of natamycin on mobility at e5 and e8 is difficult to assess due to overlap with natamycin polyene chain signal. Similarly, e14 and e17 resonances are no longer resolved and are obscured by the epoxyde carbons from natamycin (Figure 3, top).

The changes in ergosterol segmental mobility in the presence of natamycin along with the persistent spectral resolution indicate a more specific antibiotic/sterol molecular interaction, somewhat resembling that between amphotericin and ergosterol (Umegawa et al., 2012). Such molecular complexes appear to have comparatively little effect on lipid and sterol cooperative dynamics. By contrast, while the observed spectroscopic changes in amphotericin B/ergosterol spectra support the molecular model, proposed in (Umegawa et al., 2012), they also suggest that the effect is far more cooperative affecting the whole membrane, while the natamycin/ergosterol complexes appear to interact with each other and with the membrane bulk much less and, subsequently, perturb membrane lipid to much lesser extent. The significance of this observation is that while amphotericin B at a mechanistic level may affect the functionality of ergosterol-dependent proteins through significant modulation of membrane properties (te Welscher et al., 2008), natamycin appears to have a very specific effect on ergosterol, which is not transmitted to the rest of the membrane.

Besides specific interactions with sterols, followed directly from changes in ¹³C MAS NMR spectra from cholesterol and ergosterol, the addition of polyene antibiotics also affects membrane phospholipid. Within hydrated membranes, phospholipids undergo fast cooperative axial motions with GHz rates and the full width at half height (FWHH) of lipid resonances is affected by the cooperative mobility, fast axial rotation, and slow angular librations, as well as by additional, individual segmental motions. The presence of additional membrane constituents can affect any or many of these motions and the effects are seen in the high-resolution ¹³C MAS NMR spectra from the membrane lipid (Ciesielski et al., 2009; Zorin et al., 2010). In addition, the isotropic chemical shifts of each carbon environment can be affected directly by the proximity of membrane active molecules or indirectly by changes in molecular order parameters and packing (Bonev et al., 2001).

Lipid resonance full width at half height (FWHH) is affected primarily by lateral molecular librations or rocking and increases in membranes with lower bending modulus, where membrane undulation is facilitated by lose chain packing (Ciesielski et al., 2009). By contrast, the presence of cholesterol stiffens the membrane and improves packing in the chain region without impairing axial lipid rotation, which leads to significant reduction of linewidth and improved spectral resolution (Zorin et al., 2010). Resolution can also be improved under proton decoupling and mobility that interferes with decoupling can increase linewidth. Thus, the FWHH is related to the transverse relaxation rate and a good indicator of changes in librational mobility of lipids in response to membrane interactions.

The changes in DOPC ¹³C linewidth in mixed membranes with cholesterol or ergosterol before and after addition of amphotericin B or natamycin are shown in Figure 4 alongside the carbon number. Addition of amphotericin B to cholesterol containing DOPC mixed membranes leads to marked increase in linewidth, particularly in chain region, indicative of incorporation of the amphotericin B into the hydrophobic core of the membrane. The effect is consistently observed throughout chain methylenes, reflective of the cooperativity of chain motions. Broadening of carbonyl and to a lesser extent backbone and headgroup resonances arises from some degree of cooperative mobility of the whole DOPC molecule. Similar broadening of chain resonances is observed in the presence of natamycin but to a lesser extent, consistent with the comparatively less perturbing insertion of natamycin into the membrane.

Both amphotericin B and natamycin broaden DOPC resonances in ergosterol/DOPC mixed membranes. Reflective of reduced axial rotation and enhanced librational freedom, the effect is more spread out over the entire lipid molecule with a notable significant broadening of the carbonyl resonances by amphotericin B. Overall, backbone and headgroup resonances remain comparatively less perturbed, which confirms insertion of both antifungals into the membrane interior.

The presence of amphotericin B or natamycin has a minor impact on DOPC ¹³C isotropic chemical shifts in either cholesterol or ergosterol-containing membranes (Figure 5). In DOPC/cholesterol membranes natamycin addition leads to more pronounced changes in chemical shifts (Figure 5A). This is likely the result of the described earlier specific interactions with cholesterol, which lead to altered order in the DOPC component of the mixed membrane and more pronounced changes in the headgroup region. The effects on DOPC/ergosterol membranes are very similar and support the picture of collective molecular organization within the membrane, discussed earlier.

Good spectral resolution in DOPC/cholesterol membranes allows monitoring the changes in the ¹³C isotropic chemical shifts of cholesterol, as well. The presence of amphotericin B in the DOPC/cholesterol membrane has a small and comparatively uniform impact on ¹³C chemical shifts of cholesterol (Figure 6A), which reflects the observed comparatively small impact on longitudinal relaxation and linewidth (transverse relaxation). This offers further support to a cooperative and non-specific effect of amphotericin on DOPC/cholesterol membranes. The addition of natamycin to DOPC/cholesterol membranes has more location-specific impact on ¹³C chemical shifts, which correlates well with resonances proximal to the observed resonance attenuation at the sterol core, core methyls and the end of the chain (Figure 6B).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.