Characterization of the liquid-ordered state by proton MAS NMR

Abstract

We investigated if magic angle spinning (MAS) 1H NMR can be used as a tool for detection of liquid-ordered domains (rafts) in membranes. In experiments with the lipids SOPC, DOPC, DPPC, and cholesterol we demonstrated that 1H MAS NMR spectra of liquid-ordered domains (lo) are distinctly different from liquid-disordered (ld) and solid-ordered (so) membrane regions. At a MAS frequency of 10 kHz the methylene proton resonance of hydrocarbon chains in the ld phase has a linewidth of 50 Hz. The corresponding linewidth is 1 kHz for the lo phase and several kHz for the so phase. According to results of 1H NMR dipolar echo spectroscopy, the broadening of MAS resonances in the lo phase results from an increase in effective strength of intramolecular proton dipolar interactions between adjacent methylene groups, most likely because of a lower probability of gauche/trans isomerization in lo. In spectra recorded as a function of temperature, the onset of lo domain (raft) formation is seen as a sudden onset of line broadening. Formation of small domains yielded homogenously broadened resonance lines, whereas large lo domains (diameter >0.3 microm) in an ld environment resulted in superposition of the narrow resonances of the ld phase and the much broader resonances of lo. 1H MAS NMR may be applied to detection of rafts in cell membranes.

FIGURE 1

(A) Spectra of SOPC, SOPC/cholesterol 85:15 mol %, and SOPC/cholesterol 70:30 mol % at 37°C. The first order spinning sidebands are shown in the inset with 20-fold magnification. (B) Temperature-dependent changes of 10 kHz ¹H MAS NMR spectra of SOPC multilamellar vesicles. Superimposed spectra are shown to emphasize the presence of isosbestic points in the course of the gel-to-liquid crystalline phase transition. Bold lines show spectra taken at the highest (37°C) and at the lowest (2°C) temperatures. The first order spinning sidebands are shown in the inset with 20-fold magnification. (C) Temperature-dependent changes of 10 kHz ¹H MAS NMR spectra of SOPC/cholesterol 70:30 multilamellar vesicles. Superimposed spectra are shown to emphasize the presence of isosbestic points in the phase coexistence region. Bold lines show spectra taken at the highest (37°C) and at the lowest (2°C) temperatures. The first order spinning sidebands are shown in the inset with 20-fold magnification.

FIGURE 2

(A) Temperature dependence of the methylene resonance intensity at 1.3 ppm in the spinning centerband for SOPC, SOPC/cholesterol 85:15 mol %, and SOPC/cholesterol 70:30 mol %. (B) Plot of the first spinning sideband/centerband intensity ratio for SOPC, SOPC/cholesterol 85:15 mol %, SOPC/cholesterol 70:30 mol %, and DPPC/cholesterol 70:30. (C) ¹H MAS NMR spectra of SOPC, SOPC/cholesterol 85:15 mol %, SOPC/cholesterol 70:30 mol %, and DPPC/cholesterol 70:30 (from top to bottom) at the low temperature end of the transition at 4°C, −2°C, −4°C, and 35°C, respectively. The first order spinning sidebands are shown in the inset with 20-fold magnification. The SOPC spectrum corresponds to the s_o phase; SOPC/cholesterol 85:15 mol % represents coexistence of s_o and l_o, and SOPC/cholesterol 70:30 mol % is mostly the l_o phase.

FIGURE 3

(A) Deuterium NMR spectra of SOPC-d₃₅ recorded at −24°C, −14°C, −9°C, −1°C, 1°C, 2°C, 3°C, 6°C, and 26°C. (B) Deuterium NMR spectra SOPC-d₃₅/cholesterol 70:30 mol % recorded at −33°C, −23°C, −13°C, −8°C, −3°C, 2°C, 7°C, 17°C, and 27°C. (C) Temperature dependence of first moments, M₁, of deuterium NMR spectra of SOPC-d₃₅ (○), SOPC-d₃₅/cholesterol 85:15 (▵), and SOPC-d₃₅/cholesterol 70:3 (▴).

FIGURE 4

Schematic phase diagram of a SOPC-cholesterol binary mixture as discussed in the text. The lines connecting the points are guides to the eye. The following points were determined from DSC data (♦, onset; ⋄, completion of the phase transition) (43), from proton MAS NMR (▪, onset; □, completion), and from deuterium NMR (•, ○). Phase transition temperatures measured by ²H NMR on sn-1 chain deuterated SOPC were raised by 3°C to compensate for the difference in phase transition temperatures between deuterated and protonated lipids.

FIGURE 5

(A) Delay time dependence of the dipolar echo maximum for SOPC (solid symbols) and SOPC-d₃₅/cholesterol 70:30 (open symbols), measured at temperatures of 17°C (circles), 4°C, and −13°C (triangles). The curves were shifted along the y axis to reduce overlap. (B) Interpair dipolar moments determined from the slope of curves at short delay times as shown in panel A.

FIGURE 6

(A) The top panel shows the superimposed ¹H MAS NMR spectra of DOPC/DPPC 1:1 30 mol % cholesterol, recorded as a function of temperature. Spectra recorded at the highest (45°C) and the lowest (8°C) temperatures are shown as bold lines. The bottom panel shows the spectrum of the pure l_o phase at 8°C. It was generated by subtraction of a judiciously chosen fraction of the pure l_d phase spectrum, recorded at 45°C. The bold line corresponds to subtraction of 35 mol % of l_d. The thin lines reflect subtraction of smaller or larger amounts of l_d. (B) Fraction of lipid in the disordered state as a function of temperature determined from the ¹H MAS spectra (○) and the area fraction of disordered domains determined by fluorescence microscopy (▪) (S. L. Veatch and S. L. Keller, unpublished).