Macroscopic orientation

The macroscopic orientation of their extended-chain crystals depends on the orientation imparted by flow during mol ding. Because of the fibrous nature of the extended-chain crystals, these materials behave as self-reinforcing composites, with excellent mechanical properties. 

The growth of ECC under equilibrium conditions is too slow. Moreover, no macroscopic orientation appears and a structure of the type shown in Fig. 3 c is formed. Therefore this procedure cannot be used in practice. Usually, under real conditions, macroscopically oriented ECC are obtained from the melt stretched to the values of > /3cr at relatively low crystallization temperatures. Under these conditions, the formation of ECC proceeds by another mechanism. 

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. 

Abstract To understand how membrane-active peptides (MAPs) function in vivo, it is essential to obtain structural information about them in their membrane-bound state. Most biophysical approaches rely on the use of bilayers prepared from synthetic phospholipids, i.e. artificial model membranes. A particularly successful structural method is solid-state NMR, which makes use of macroscopically oriented lipid bilayers to study selectively isotope-labelled peptides. Native biomembranes, however, have a far more complex lipid composition and a significant non-lipidic content (protein and carbohydrate). Model membranes, therefore, are not really adequate to address questions concerning for example the selectivity of these membranolytic peptides against prokaryotic vs eukaryotic cells, their varying activities against different bacterial strains, or other related biological issues. 

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. 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...

Fig. 8 Representative solid-state 19F-NMR spectra of 19F-labelled gramicidin S (substituted at both Leu positions with 4F-Phg), embedded in macroscopically oriented ghost membranes (left column) and DMPC bilayers (right column) at a peptide-to-lipid ratio of about 1 40 [in (F) the lipid was 1 1 DMPC/cholesterol]. Depending on temperature, the peptide can assume different alignment states, which are strikingly similar in the native membranes and the model bilayers...

Grobner G, Taylor A, Williamson PT, Choi G, Glaubitz C, Watts JA, de Grip WJ, Watts A (1997) Macroscopic orientation of natural and model membranes for structural studies. Anal Biochem 254 132-138... 

Surface reconstruction or adsorption can often cause a vicinal surface with a single macroscopic orientation to facet into surfaces with different orientations. Generally the reconstruction occurs on a particular low-index flat face, and lowers its free energy relative to that of an unreconstructed surface with the same orientation. However the same reconstruction that produces the lower free energy for the flat face generally increases the energy of surface distortions such as steps that disturb the reconstmction. Thus reconstmction is often observed only on terraces wider than some critical terrace width Wc. When steps are uniformly distributed initially and if Wc is much greater... 

Rusakov 107 108) recently proposed a simple model of a nematic network in which the chains between crosslinks are approximated by persistent threads. Orientional intermolecular interactions are taken into account using the mean field approximation and the deformation behaviour of the network is described in terms of the Gaussian statistical theory of rubber elasticity. Making use of the methods of statistical physics, the stress-strain equations of the network with its macroscopic orientation are obtained. The theory predicts a number of effects which should accompany deformation of nematic networks such as the temperature-induced orientational phase transitions. The transition is affected by the intermolecular interaction, the rigidity of macromolecules and the degree of crosslinking of the network. The transition into the liquid crystalline state is accompanied by appearence of internal stresses at constant strain or spontaneous elongation at constant force. 

Theory for the self- and tracer-diffusion of a diblock copolymer in a weakly ordered lamellar phase was developed by Fredrickson and Milner (1990). They modelled the interactions between the matrix chains and a labelled tracer molecule as a static, sinusoidal, chemical potential field and considered the Brownian dynamics of the tracer for small-amplitude fields. For a macroscopically-oriented lamellar phase, they were able to account for the anisotropy of the tracer diffusion observed experimentally. The diffusion parallel and perpendicular to the lamellae was found to be sensitive to the mechanism assumed for the Brownian dynamics of the tracer. If the tracer has sufficiently low molecular weight to be unentangled with the matrix, then its motion can be described by a Rouse model, with an added term representing the periodic potential (Fredrickson and Bates 1996) (see Fig. 2.50). In this case, motion parallel to the lamellae does not change the potential on the chains, and Dy is unaffected by... 

The requirement for a surface of a given macroscopic orientation B to break up into two new surfaces of orientation nA and nc is that the total surface energy be reduced. 

Chien and Cada [42] have prepared optically active and photoactive SCLC copolymers, 15, with the 4-alkoxyphenyl-4 -alkoxycinnamate chromophore, with the intention of creating LC polysiloxane networks that could be used to prepare macroscopically oriented organic ferroelectric polymers for electro-optical devices. Optical activity was introduced into the polymer by the use of a chiral spacer. Those copolymers which were mesogenic exhibited properties characteristic of a Sc. phase. UV-irradiation of thin films of the polymers in their mesomorphic states at 90°C, led to a loss of the IR absorption at 1635 cm-1 that is due to the cinnamate double bond, and to cross-linking. Long-term irradiation led to... 

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