Lipid bilayers magnetically oriented

Figure 18.7 3,P NMR spectra of MSI-103 in DMPC. (a) P/L= 1 200, nonoriented multilamellar vesicles sample. The broad signal shows the typical powder spectrum of a liquid crystalline lamellar lipid phase, (b) P/L = 1 20, macroscopically oriented sample. The peak around 28 ppm shows that the sample is well oriented with the lipid bilayer normal oriented parallel to the magnetic field.

Because the lipid bilayer is oriented with respect to the magnetic field with the bilayer surface parallel to the magnetic field and the tt-helical axis of melittin... 

Several works have been reported for macroscopically orientated biological membranes.106-109 The biomembrane alignment can be carried out mechanically or magnetically. The first one relies on the deposition of lipid bilayers on the surface of a rigid support (glass plates) such that the bilayer normal is perpendicular to the surface of the support itself. Small peptides and the lipid bilayers can be dissolved in organic solvents which are successively removed under vacuum.105 The re-hydration of the system in a chamber of an optimized temperature, humidity and time gives rise to the desired orientation. 

Fig. 1 Solid-state NMR structure analysis relies on the 19F-labelled peptides being uniformly embedded in a macroscopically oriented membrane sample, (a) The angle (0) of the 19F-labelled group (e.g. a CF3-moiety) on the peptide backbone (shown here as a cylinder) relative to the static magnetic field is directly reflected in the NMR parameter measured (e.g. DD, see Fig. 2c). (b) The value of the experimental NMR parameter varies along the peptide sequence with a periodicity that is characteristic for distinct peptide conformations, (c) From such wave plot the alignment of the peptide with respect to the lipid bilayer normal (n) can then be evaluated in terms of its tilt angle (x) and azimuthal rotation (p). Whole-body wobbling can be described by an order parameter, S rtlo. (d) The combined data from several individual 19F-labelled peptide analogues thus yields a 3D structural model of the peptide and how it is oriented in the lipid bilayer...

Fig. 5 Membrane models for NMR structure analysis, (a) An isotropic detergent micelle (left) is compared to the dimensions of lipid bilayers (right), (b) Macroscopically oriented membrane samples can be prepared on solid support, as nanodiscs, or as magnetically oriented bicelles. (c) Nomenclature and variability of liposomes small (SUV, 20-40 nm), intermediate (IUV, 40-60 nm), large (LUV, 100-400 nm), and giant unilamellar vesicles (GUV, 1 pm) multi-lamellar (MLV), oligo-lamellar (OLV) and highly heterogeneous multi-oligo-lamellar vesicles (MOLV)...

P SS NMR data of 14-mer amphipathic peptide (composed of leucines and phenylalanines modified by crown ethers) embedded into membranes were reported by Ouellet et al.125 The preliminary study indicated that the 14-mer peptide remains at the surface of bilayers and perturbs the lipid orientation relative to the magnetic field. 15N-31P REDOR experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the PL headgroup and polar region of DMPC MLVs. The results strongly suggest that the 14-mer peptide destabilises the lipid bilayer via the induction of a positive curvature strain. 

As an altemative approach to MAS experiments on immobilized proteins, membrane proteins may be incorporated into planar lipid bilayers, which may be uniaxially oriented with the bi layer normal parallel to the external magnetic field. This implies that the sample will display single-crystal like spectra and hence sample spinning is not needed to provide high resolution. In this section, we will numerically investigate some of the fundamental aspects one needs to consider when performing experiments on uniaxially oriented membrane proteins. 

Fig. 33. Directions of the principal axes of the l3C chemical shift tensor of the C=0 group, helical axis, and static magnetic field, B0, and 13C NMR spectral patterns of the C=0 carbons corresponding to the orientation of the a-helix with respect to the surface of the magnetically oriented lipid bilayers. Simulated spectra were calculated using 5, =241 ppm, 22 =189 ppm, and < 33 =96 ppm for the rigid case (a), rotation about the helical axis (slow MAS) (b), fast MAS (c), magnetic orientation parallel to the magnetic field (d), an angle 8 with the magnetic field (e), and the direction perpendicular to the magnetic field (f).11 Reproduced with permission from the Biophysical Society.

Fig. 34. (a) Schematic representation of the orientation of melittin helices bound to magnetically oriented lipid bilayers. N and C terminal helix axes make angles of 30 and 10° respectively with the average axis, which is perpendicular to the bilayer surface. Two kink angles (140 and 160°) can be taken, but they cannot be distinguished by this NMR experiment. (b) The lytic process of lipid bilayers in the presence of melittin at temperatures below Tm. O,, lipid and melittin molecules respectively." Reproduced with permission from the Biophysical Society. 

Abdine et in their review, stated the importance of the development of cell-free expression systems to study, using SSNMR, the structure of membrane proteins in their native environment and the hydrated lipid bilayer. Koch et al. pointed to the usefulness of solid-state F-NMR to study the structures of peptides in native membranes of defined orientation, while Mote et al. ° suggested the use, for the same purpose, of NMR and magnetically aligned lipid bilayers in order to determine the structure and topology of larger integral membrane proteins. 

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