Biomembranes, native

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... 

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

Given the overwhelming compositional complexity of biomembranes, a slightly better representation can be achieved when lipid extracts from native sources are used... 

A similar difference has been observed for Kp M values of the anticancer drags tamoxifen and 4-hydroxytamoxifen determined in mitochondria, sarcoplasmic reticulum, and in liposomes made from lipids extracted from the corresponding native membranes [85]. The largest Kp M was observed in mitochondria, followed by sarcoplasmic reticulum and liposomes. The authors introduced for these experiments derivative spectroscopy, a reliable and rapid procedure to estimate drag partitioning into biomembranes. The method used the shift in the absorption spectra of the drug when removed from the aqueous phase to a hydrophobic environment (see Section 3.10). 

Often, experimental studies of lipid systems are based on spectroscopic approaches, which in turn frequently employ probes for enhancement of sensitivity and resolution. For example, in NMR, hydrogen atoms of lipids are replaced with deuterium, and in fluorescence spectroscopy and imaging, native lipid molecules are replaced with lipids in which one of the hydrocarbon chains is linked covalently to a fluorescent marker such as pyrene or diphenylhexatriene. Fluorescent markers allow one to follow numerous cellular processes in real time, such as intracellular trafficking of molecules and formation of domains within a biomembrane, see Fig. 3. The downside is that the probes tend to perturb their environment and affect the thermodynamic state of the system. Experiments have shown, for example, that probes may change the main transition temperature of a lipid membrane, and that the dynamics of probes may deviate considerably from the dynamics of corresponding native molecules (see discussion in Reference 27). Therefore, we wish to pose several questions. What is the range of perturbations induced by the probe How significant are these perturbations actually ... 

Because of its occurence in diseased tissue, the mode of association of cholesterol esters with biomembranes is of interest. Possible modes of association could be droplets within the hydrophobic core of the membrane bilayer, binding to membrane protein or as part of membrane attached serum lipoproteins. A potentially useful model system for investigating this association is the membrane of the microorganism Mycoplasma capricolum. The Mycoplasma due to their simplicity have served as model membrane systems in many studies. As mentioned previously, cholesterol esters show complex behavior that is a function of thermal history, impurities and physical packing constraints. Using DSC on native membranes and extracted membrane material, it was possible to demonstrate that the majority of cholesterol esters associated with the membranes of M. capricolum exist as relatively large and pure liquid droplets (17). 

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