Samples oriented lipid bilayers

For the orientation-based structure analysis of MAPs, uniformly oriented lipid bilayers are typically prepared on solid supports as illustrated in Fig. 2 [23, 47, 55]. These mechanically oriented membranes are advantageous for static ssNMR experiments, as they provide a robust way to orient a sample with any desired lipid composition, peptide concentration, and at any desired temperature. The lipids... 

In order to establish structural constraints on proteins and peptides from solid-state NMR, it is important to consider all aspects from appropriate labeling of the sample, selection of the experiments providing the desired information, and to have appropriate reference data available to allow extraction of structural data from the (anisotropic interaction) parameters determined by the experiment. As an example. Cross and co-workers investigated the conformation of the ion channel gramicidin A using selectively N-labeled peptides in uniaxiaUy oriented lipid bilayers c.f.. Section 4.2). To translate the measured " N chemical shifts in the oriented samples into stractural constraints, it is necessary to determine the magnitude and orientation of the " N chemical shift tensors... 

Fig. 6.4.4. N spectra obtained at 20.3 MHz of labeled gramicidin A from different sample preparations in oriented lipid bilayers. (A) deuterium exchange of the indole proton in Trpy] gramicidin A results in the observed dipolar triplet centred on the observed (B)...

At this point, we mention a related class of sample, namely oriented samples. In the case of a perfect macroscopic ordering, each equivalent nucleus is oriented identically, and the situation is the same as that in a single crystal. Specific oriented samples of relevance (with varying degrees of ordering) include polymer fibres [4], liquid crystals (LC), [12, 13] and membrane proteins in oriented lipid bilayers [14]. We will return to the latter two cases in the discussion of two-dimensional experiments in Sections 9.5 and 9.6. 

A home-built solid state NMR probe for membrane protein studies has been described by Kim et al. Proteins in highly oriented lipid bilayer samples are useful to study membrane protein structure determination. Planar lipid bilayers aligned and supported on glass slide were prepared. The stack of glass slide with planar lipid bilayers is not well fit for commercial solid state NMR probe with round coil. Therefore, homebuilt solid state NMR probe was built by Kim et al. The overall filling factor of the coil was much better and the large surface area increased the extent to orientation by providing uniform environments for the... 

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. 2 Mechanically oriented bilayer samples as a membrane model for ssNMR. (a) Illustration of the hydrated lipid bilayers with MAPs embedded, the glass supports, and the insulating wrapping, (b) A real sample consists of 15 stacked glass slides, (c) Schematic solid-state 19F-NMR lineshapes from an oriented CF3-labelled peptide (red), and the corresponding powder lineshape from a non-oriented sample (grey), (d) Illustration of typical orientational defects in real samples - the sources of powder contribution in the spectra...

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)...

The exact dimensions of a phospholipid bilayer membrane in terms of the in-plane area and the height of the lipid molecules as well as the thickness of the water layer that is associated with them is dependent on the chemical identity of the phospholipid head group, the length and the degree of saturation of the acyl chains, and the degree of hydration. This information may be obtained from a combination of small-angle X-ray diffraction by MLV or oriented multi-bilayer samples of phospholipids in excess water, electron and/or neutron density profiles across lipid bilayers, and atomic level molecular dynamics simulations of hydrated lipid bilayers. H-NMR studies on selectively deuter-ated phospholipids have also been important in elucidating acyl... 

To obtain orientational constraints, peptide/lipid samples for 19F NMR studies are usually prepared with lipid bilayers that are macroscopically oriented on glass plates [52-54], Lipids and peptides in appropriate amounts are co-dissolved in organic solvent and spread onto thin glass plates. After removal of the solvent, the plates are stacked and placed in a... 

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.

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. 

The high-resolution and the high scaling factor of 2D PISEMA, for the first time, enabled the use of the dipolar dimension to resolve resonances from non-selectively or uniformly labeled proteins. Three-Dimensional experiments were used to enhance the resolution of resonances from uniformly N-labeled peptides and proteins embedded in lipid bilayers. This was successfully demonstrated on aligned samples containing uniformly N-labeled membrane-associated peptides and proteins. Two-dimensional PISEMA spectra of some of these systems showed limited resolution due to a small frequency dispersion of resonances from a-helices oriented on the surface of the bilayer in both N chemical shift and H- N dipolar coupling dimensions. However, when an additional H chemical shift dimension was invoked, the 3D H chemical shift/ H- N dipolar coupling/ N chemical shift spectra of these systems considerably increased the resolution of peaks. ... 

The transmembrane portion of the M2 protein from the influenza A virus has been studied in hydrated DMPC lipid bilayers with solid-state NMR.209 Orientational constraints are obtained from the isotopically labeled peptide samples mechanically aligned between thin glass plates. I5N chemical shifts from single-site labeled samples constrain the molecular frame with respect to... 

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