Determining the effects of lipophilic drugs on membrane structure by solid-state NMR spectroscopy: the case of the antioxidant curcumin

Abstract

Curcumin is the active ingredient of turmeric powder, a natural spice used for generations in traditional medicines. Curcumin's broad spectrum of antioxidant, anticarcinogenic, antimutagenic, and anti-inflammatory properties makes it particularly interesting for the development of pharmaceutical compounds. Because of curcumin's various effects on the function of numerous unrelated membrane proteins, it has been suggested that it affects the properties of the bilayer itself. However, a detailed atomic-level study of the interaction of curcumin with membranes has not been attempted. A combination of solid-state NMR and differential scanning calorimetry experiments shows curcumin has a strong effect on membrane structure at low concentrations. Curcumin inserts deep into the membrane in a transbilayer orientation, anchored by hydrogen bonding to the phosphate group of lipids in a manner analogous to cholesterol. Like cholesterol, curcumin induces segmental ordering in the membrane. Analysis of the concentration dependence of the order parameter profile derived from NMR results suggests curcumin forms higher order oligomeric structures in the membrane that span and likely thin the bilayer. Curcumin promotes the formation of the highly curved inverted hexagonal phase, which may influence exocytotic and membrane fusion processes within the cell. The experiments outlined here show promise for understanding the action of other drugs such as capsaicin in which drug-induced alterations of membrane structure have strong pharmacological effects.

Figure 1

Keto-enol form of curcumin, the dominant tatuomer of curcumin. The keto-enol from is stabilized by an intramolecular hydrogen bond, shown here by a dashed red line.

Figure 2. Curcumin lowers the enthalpy and phase transition temperature of DMPC and DPPC bilayers

DSC heating scans for (A) DMPC and (B) DPPC with varying concentrations of curcumin show that it lowers the temperature of the DMPC bilayer phase transition (from gel to liquid crystalline phase) and decreases the enthalpy of main phase transition, making the gel phase more fluid and the liquid crystalline phase more rigid. All curcumin values are given as mole percentages with a constant lipid concentration of 2.95 mM.

Figure 3. Curcumin promotes the formation of the negatively curved inverted hexagonal (H_II) phase

DSC heating scans of DiPoPE containing the listed molar percentage of curcumin.

Figure 4. Changes in the ¹⁴N quadrupole coupling of magnetically aligned 9:2 DMPC:DHPC bicelles containing the listed molar concentration of curcumin show that curcumin increases the asymmetry around the ¹⁴N nucleus in the choline group

The inner peaks are associated with DHPC and the larger, outer peaks are associated with DMPC. The vertical lines are for guide purposes only.

Figure 5

Top: A 1D ¹³C chemical shift and a 2D PDLF spectrum of magnetically aligned 9:2 DMPC:DHPC bicelles (pH 6.0) in the absence of curcumin. Right: Molecular structure of DMPC showing the numbering convention used. Bottom: C-H dipolar coupling spectral slices extracted from 2D PDLF spectra of magnetically aligned bicelles without (bottom left) and with 5 mole % curcumin (bottom right).

Figure 6. The molecular order parameters determined by 2D PDLF experminents show site-specific ordering of DMPC in DMPC/DHPC bicelles by curcumin

Curcumin induced overall ordering of the bilayer at all concentrations tested. A. Order parameter profile in the lipid headgroup region (carbons γ -α). The non-monotonic response is indicative of a conformational change of the lipid headgroup in the presence of curcumin. B. Order parameter profile in the lipid headgroup region (carbons g3–3). The ordering effect is strongest in this region. C. Order parameter profile in the hydrophobic core of the membrane (carbons 4–11). The terminal carbons are more disordered than the pure bicelle sample at the lower concentrations of curcumin but are more ordered at higher concentrations (1–5%). D. Cartoon schematic of the physical changes induced by curcumin in the membrane. Curcumin at low molar concentrations is monomeric and oriented with its long axis along the membrane normal. Curcumin is effective at these concentrations in ordering the membrane except for the terminal carbons below the curcumin molecule. At higher concentrations, curcumin oligomerizes in the membrane, reducing its thickness and molecular order relative to the samples with lower concentrations of curcumin.