Solid-state NMR studies of magnetically aligned phospholipid membranes: taming lanthanides for membrane protein studies

Abstract

The addition of lanthanides (Tm3+, Yb3+, Er3+, or Eu3+) to a solution of long-chain phospholipids such as dimyristoylphosphatidylcholine (DMPC) and short-chain phospholipids such as dihexanoylphosphatidylcholine (DHPC) is known to result in a bilayer phase in which the average bilayer normal aligns parallel to an applied magnetic field. Lanthanide-doped bilayers have enormous potential for the study of membrane proteins by solid-state NMR, low-angle diffraction, and a variety of optical spectroscopic techniques. However, the addition of lanthanides poses certain challenges to the NMR spectroscopist: coexistence of an isotropic phase and hysteresis effects, direct binding of the paramagnetic ion to the peptide or protein of interest, and severe paramagnetic shifts and line broadening. Lower water concentrations and larger DMPC/DHPC ratios than those typically used in bicelles consistently yield a single oriented bilayer phase that is stable over a wide range of temperature (approximately 35-90 degrees C). Among the above choice of lanthanides, Yb3+ is found to give minimal paramagnetic shifts and line broadening at acceptably low concentrations necessary for alignment (i.e., Yb3+/DMPC mole ratios equal to or greater than 0.01). Finally, the addition of a phospholipid chelate, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine--diethylenetriaminepent aacetic acid, is observed to significantly reduce paramagnetic broadening and presumably prevent direct association of the peptide with the lanthanide ions.

On the magnetic anisotropy of lanthanide-containing metallomesogens

“…For instance, if ⌬ is negative for the first group of lanthanide compounds, ⌬ is positive for the second group and vice versa. All experimental results obtained for lanthanide-containing liquid crystals 13,14 and for phospholipid bilayers [15][16][17][18][19] doped with lanthanide chelates are in agreement with a negative ⌬ value for the first group and a positive ⌬ value for the second group. But as we will show further in this paper, the reverse situation can be expected too.…”

Investigating magnetically aligned phospholipid bilayers with EPR spectroscopy at Q-band (35GHz): optimization and comparison with X-band (9GHz)

“…A recently developed model membrane system called magnetically aligned phospholipid bilayers (bicelles), [1,2,6,7] that spontaneously align in a magnetic field, has been demonstrated to be successful for nuclear magnetic resonance (NMR) spectroscopic studies of membrane and integral membrane proteins [2,3,[7][8][9][10][11][12][13][14]. Generally, bicelles consist of a binary mixture of a long chain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and short chain 1,2-dihexanoylsn-glycero-3-phosphocholine (DHPC) phospholipids [2,15].…”

Application of Weakly Oriented Non‐ionic Liquid Crystal Solvents to NMR Studies of Poly‐ethylene Oxide (PEO)

“…Therefore, a technique originally proposed four decades ago 1,2 was recently revisited and found surprisingly to have important applications in protein NMR spectroscopy. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] The essence of this technique is that a tiny residual dipolar coupling (from a few to hundreds of hertz) between two spins can be reintroduced by rendering the NMR solvent weakly anisotropic. Particularly, the dipolar couplings between spins which otherwise are averaged out by isotropic molecular motion of both the solvent and solute molecules can be slightly restored.…”

“…The studies hitherto carried out in various research groups have been focused on proteins. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Structurally, proteins are heterogeneous polymers and consequently are anticipated to be more susceptible to the weak anisotropy of solvents. It is also suspected that the weak anisotropy in susceptibility (or equivalently residual dipolar and/or quadrupolar couplings) may partially arise from the large-scale heterogeneity, i.e., tertiary or higher orders of structure, of the biological macromolecules.…”