High-resolution 2D NMR spectroscopy of bicelles to measure the membrane interaction of ligands

Abstract

Magnetically aligned bicelles are increasingly being used as model membranes in solution- and solid-state NMR studies of the structure, dynamics, topology, and interaction of membrane-associated peptides and proteins. These studies commonly utilize the PISEMA pulse sequence to measure dipolar coupling and chemical shift, the two key parameters used in subsequent structural analysis. In the present study, we demonstrate that the PISEMA and other rotating-frame pulse sequences are not suitable for the measurement of long-range heteronuclear dipolar couplings, and that they provide inaccurate values when multiple protons are coupled to a 13C nucleus. Furthermore, we demonstrate that a laboratory-frame separated-local-field experiment is capable of overcoming these difficulties in magnetically aligned bicelles. An extension of this approach to accurately measure 13C-31P and 1H-31P couplings from phospholipids, which are useful to understand the interaction of molecules with the membrane, is also described. In these 2D experiments, natural abundance 13C was observed from bicelles containing DMPC and DHPC lipid molecules. As a first application, these solid-state NMR approaches were utilized to probe the membrane interaction of an antidepressant molecule, desipramine, and its location in the membrane.

Fig. 1

³¹P NMR spectrum obtained by acquiring the FID after a 90° RF pulse in the presence of proton decoupling. 64 transients were accumulated. The chemical shift scale was referenced by setting the isotropic chemical shift peak observed at 10°C to 0 ppm.

Fig. 2

Experimental protocols of 2D experiments used for the measurements of heteronuclear dipolar couplings and the corresponding simulated dipolar coupling spectra for a three spin system CH₂ with d_CH(1) = 20 kHz and d_CH(2) = 4 kHz. (a) SLF; (b) PDLF; (c) Rotating-frame SLF.

Fig. 3

(a) A 2D PDLF spectrum of DMPC/DHPC bicelles at 37°C. 64 scans were accumulated for each of 200 points in the t₁ dimension with an increment time of 384 μs. The contact time for the CP transfer was set to 3.0 ms. Proton RF field during the t₁ evolution corresponded to 31 kHz. A 1D ¹³C chemical shift spectrum is shown at the top with assignments of peaks to individual carbons of the DMPC molecule. (b) Part of the PDLF spectrum in DMPC/DHPC bicelles at 50°C demonstrating the increased dipolar resolution in the crowded spectra range between 31 and 34 ppm. Corresponding part of the PDLF spectrum obtained at 37°C is also shown for comparison.

Fig. 4

Representative slices along the dipolar coupling dimensions from SLF, PDLF and PISEMA spectra. Frequency axes were corrected for the respective dipolar scaling factors k = 0.42 (SLF and PDLF) and 0.79 (PISEMA).

Fig. 5

Simulated (dashed lines) and experimental (solid lines) ¹H-¹³C dipolar coupling spectra corresponding to C2 and β carbons of the DMPC molecule obtained using SLF, PDLF and PISEMA pulse sequences. Only couplings to directly bonded protons are accounted for in the simulations. Frequency axes were corrected for the respective dipolar coupling scaling factors k = 0.42 (SLF and PDLF) and 0.79 (PISEMA).

Fig. 6

Contour plot of the headgroup and glycerol region of the 2D ¹³C-¹H correlation spectrum (top). The tilted dipolar doublets are indicated by solid lines. The doublets are due to ¹³C-³¹P and ¹H-³¹P dipolar interactions resulting in splittings of the ¹³C-¹H cross peaks along the horizontal axis and vertical axis, respectively. The experimental conditions are as mentioned for the PDLF spectrum in Fig. 3. Comparison to the ¹³C-¹H PDLF spectrum (bottom) was used to assign the peaks.

Fig. 7

Cross sections from the 2D ³¹P-¹H PDLF spectrum at the chemical shift of the ³¹P resonance in DMPC and DHPC molecules. 64 transients were accumulated for each of 32 points in the t₁ dimension with an increment time of 2300 μs. The location of a desipramine molecule relative to a DMPC lipid in bicelles. A change in the head group conformation of DMPC is also indicated. The relevant hydrogen atoms are included in the figure, while all other hydrogen atoms are not represented.

Fig. 8

Molecular structures of desipramine (left) and DMPC (right).