Oriented membrane models

Keywords Solid-state NMR structure analysis 19F-labeling Membrane-active peptides Native biomembranes Oriented membrane models Antimicrobial peptides... 

Hollars C W and Dunn R C 2000 Probing single molecule orientations in model lipid membranes with near-field scanning optical microscopy J. Phys. Chem 112 7822-30... 

Here, we discuss a solid-state 19F-NMR approach that has been developed for structural studies of MAPs in lipid bilayers, and how this can be translated to measurements in native biomembranes. We review the essentials of the methodology and discuss key objectives in the practice of 19F-labelling of peptides. Furthermore, the preparation of macroscopically oriented biomembranes on solid supports is discussed in the context of other membrane models. Two native biomembrane systems are presented as examples human erythrocyte ghosts as representatives of eukaryotic cell membranes, and protoplasts from Micrococcus luteus as membranes... 

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)...

Compared to the sizes of living cells,1 IUV and LUV resemble the dimensions of enveloped viruses (ranging from 80 to 400 nm) [94], while GUVs resemble typical bacteria and erythrocytes (1-7 pm). Eukaryotic cells tend to be even larger (10-30 pm in animals, 10-100 pm in plants). These dimensions imply that, except for viruses or specific sub-cellular membranes, flat bilayers are the only relevant membrane models. Hence, macroscopically oriented bilayers on solid supports (see... 

Fig. 7 Comparison of the solid-state 31P-NMR spectra from erythrocyte ghosts (left column) and of DMPC model membranes (right column). Samples are prepared as a non-oriented suspension in excess water (a, d), and as macroscopically oriented membranes on glass slides that are aligned either parallel (b, e) or perpendicular (c, f) to the static magnetic field...

Natural biological membranes consist of lipid bilayers, which typically comprise a complex mixture of phospholipids and sterol, along with embedded or surface associated proteins. The sterol cholesterol is an important component of animal cell membranes, which may consist of up to 50 mol% cholesterol. As cholesterol can significantly modify the bilayer physical properties, such as acyl-chain orientational order, model membranes containing cholesterol have been studied extensively. Spectroscopic and diffraction experiments reveal that cholesterol in a lipid-crystalline bilayer increases the orientational order of the lipid acyl-chains without substantially restricting the mobility of the lipid molecules. Cholesterol thickens a liquid-crystalline bilayer and increases the packing density of lipid acyl-chains in the plane of the bilayer in a way that has been referred to as a condensing effect. 

In all three cases amphiphiles orient spontaneously to form structures resembling the phospholipid arrangement in biomembranes. These membrane models allow a variety of investigations of physical membrane properties which could not be conducted with the complex natural systems. 

Zeolite single crystals may serve as zeolite membrane models. We mention the early elegant work of Hayhurst and Paravar [10] on an oriented large silicalite-1 crystal embedded in epoxy resin. 

Norden, B., Lindblom, G. and Jonas, I. (1977). Linear dichroism spectroscopy as a tool for studying molecular orientation in model membrane systems. Journal of Physical Chemistry, 81, 2086-93. 

The elastic membrane model, formulated in terms of elastic moduli and u r), provides a significantly reduced description of insertion phenomena. More detailed analysis should account for the orientation and displacement of the lipid molecules as well as some of their internal degrees of freedom. A step in this direction has been made, for instance, in Ref. 95. At short-length scales and near nonuniformities, lipid molecules cannot attain the normal orientations typical of their mean behavior on a macroscopic scale, which must inevitably affect their elastic properties. More detailed statistical mechanical analysis and simulational studies might provide useful insight into such behavior. 

Antimicrobial peptides, isolated from the dorsal glands of Australian tree frogs, have been studied using multinuclear solid state NMR. Specifically N-labelled peptides were studied using solid state NMR to determine their structure and orientation in model lipid bilayers. The effect of these peptides on phospholipid membranes was determined by and P solid state NMR... 

For structures with a high curvature (e.g., small micelles) or situations where orientational interactions become important (e.g., the gel phase of a membrane) lattice-based models might be inappropriate. Off-lattice models for amphiphiles, which are quite similar to their counterparts in polymeric systems, have been used to study the self-assembly into micelles [ ], or to explore the phase behaviour of Langmuir monolayers [ ] and bilayers. In those systems, various phases with a nematic ordering of the hydrophobic tails occur. 

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