How lipid headgroups sense the membrane environment: an application of ¹⁴N NMR

Abstract

The orientation of lipid headgroups may serve as a powerful sensor of electrostatic interactions in membranes. As shown previously by (2)H NMR measurements, the headgroup of phosphatidylcholine (PC) behaves like an electrometer and varies its orientation according to the membrane surface charge. Here, we explored the use of solid-state (14)N NMR as a relatively simple and label-free method to study the orientation of the PC headgroup in model membrane systems of varying composition. We found that (14)N NMR is sufficiently sensitive to detect small changes in headgroup orientation upon introduction of positively and negatively charged lipids and we developed an approach to directly convert the (14)N quadrupolar splittings into an average orientation of the PC polar headgroup. Our results show that inclusion of cholesterol or mixing of lipids with different length acyl chains does not significantly affect the orientation of the PC headgroup. In contrast, measurements with cationic (KALP), neutral (Ac-KALP), and pH-sensitive (HALP) transmembrane peptides show very systematic changes in headgroup orientation, depending on the amount of charge in the peptide side chains and on their precise localization at the interface, as modulated by varying the extent of hydrophobic peptide/lipid mismatch. Finally, our measurements suggest an unexpectedly strong preferential enrichment of the anionic lipid phosphatidylglycerol around the cationic KALP peptide in ternary mixtures with PC. We believe that these results are important for understanding protein/lipid interactions and that they may help parametrization of membrane properties in computational studies.

Figure 1

Schematic representation of the angles θ, φ, and δ where φ is the PN direction and δ is the angle between the CN and PN vectors of the PC headgroup. The γ- methyl groups of the choline are removed for clarity of the angle definitions. For details see text.

Figure 2

Fit of Eq. 1 to the calculated P-N angles of the lipid headgroup with the membrane surface from Semchyschyn and Macdonald (33) and the corresponding values of the ¹⁴N quadrupolar splittings in this study.

Figure 3

(A) Representative ³¹P NMR spectra (left column) and ¹⁴N NMR spectra (right column) of a dispersion of 30 μmol of lipids at 40°C. The asterisks in the right-hand top spectrum indicate the splittings from the quaternary ammonium of DMTAP. This assignment was confirmed by the integral ratio of the fitted intensities of the two components when varying the DMPC/DMTAP ratio. (B) Choline ¹⁴N quadrupole splittings (y axis, left) as a function of charged amphiphile fraction (solid symbols). The charge density is negative in the presence of DMPG (squares), positive in the presence of DMTAP (circles), and zero for pure DMPC (diamonds). The open symbols represent the corresponding average angles (y axis, right) with the bilayer plane of the P-N vector calculated using the model and parameters described in the Material and Methods. The inset schematically represents the behavior of the polar headgroup as determined by Sherer and Seelig (8) or Semchyschyn and Macdonald (33). The γ-methyl groups of the choline are removed for clarity.

Figure 4

Effect of temperature on various lipid mixtures. Top panel: From top to bottom, ¹⁴N quadrupolar splittings of DMPC with 20 mole % DMPG (squares), DMPC (diamonds), DMPC with 5, 10, 15, 20 mole % DMTAP (circles). Lower panel: ¹⁴N quadrupolar splitting of various dispersions of PC with different acyl chains, of a binary mixture of DMPC/Cholesterol 8:2 and of DPPC/DLPC 8:2. N.B. DPPC is depicted only from 40—C.

Figure 5

Effect of peptides on the choline head orientation at 40°C. (A) Effect of KALP23 (squares) and its neutral version Ac-KALP23 (triangles), compared to DMTAP (circles). (B) Effect of HALP23 at pH9 (open diamonds) and pH4 (solid diamonds).

Figure 6

Effect of hydrophobic mismatch on the orientation of the choline head. Left panel: Effect of 3 mole % KALP peptides (squares) on DMPC (line) at 40°C as a function of peptide hydrophobic length. The triangle represents the effect of 3 mole % Ac-KALP23. Right panel: Effect of 2 mole % (diamonds), 3 mole % (squares) of KALP23, and 3 mole % Ac-KALP23 (triangles) on the headgroup orientation of varying cis-unsaturated PC lipids (circles) at 40°C.

Figure 7

(A) Effect of peptides on PC ¹⁴N quadrupolar splittings in mixed DMPC/DMPG bilayers at 40°C: lipid bilayer without peptides (circles); Ac-KALP23 at peptide/lipid ratio (p/l) = 2 mole % (triangles); KALP23 at p/l = 1 mole % (diamonds); and p/l = 2 mole % (squares). Open and closed symbols represent two series of experiments using different batches of lipids and peptide and using a different spectrometer console. (B) Orientation of the P-N vector with respect to the bilayer plane as a function of DMPG in DMPC: lipid only (circles), with 2 mol % Ac-KALP23 (triangles), 1 mole % KALP23 (diamonds), and 2 mole % KALP23 (squares). (C) Same as B except that a fictive % DMPG offset is used (14 lipid mole % and 28 lipid mole % for 1 and 2 mole % of KALP23, respectively).