Lipopeptides

B. anthracis and related species.41,44 6 Some of these peaks have been identified (e.g., as small acid soluble spore proteins and cyclic lipopeptides), but others remain uncharacterized. There is no agreement among different laboratories as to which markers are suitable for chemotaxonomic differentiata-tion of species (i.e., are consistently found in one species versus another) or for strain identification (i.e., are reproducibly found in one strain but not another). Further, although it might be anticipated that surface proteins can be preferentially ionized or extracted, the ultra-structural origin of some peptides within the cell is not always clear. 

Madonna, A. J. Voorhees, K. J. Tarenko, N. I. Laiko, V. V. Doroshenko, V. M. Detection of cychc lipopeptide biomarkers from Bacillus species using atmospheric... 

Baltz, R.H., Miao, V. and Wrigley, S.K. (2005) Natural products to drugs daptomycin and related lipopeptide antibiotics. Natural Product Reports, 22, 717. 

Miao, V., Brost, R., Chappie, J. et al. (2006) The lipopeptide antibiotic A54145 biosynthetic gene cluster from Streptomyces fradiae. Journal of Industrial Microbiology Biotechnology, 33, 129. 

Muller, C., Nolden, S., Gebhardt, P. et al. (2007) Sequencing and analysis of the biosynthetic gene cluster of the lipopeptide antibiotic friulimicin in Aclinoplanesfriuliensis. Antimicrobial Agents and Chemotherapy, 51, 1028. 

Coeffet-Le Gal, M.F., Thurston, L., Rich, P. et al. (2006) Complementation of daptomycin dpt A and dptD deletion mutations in trans and production of hybrid lipopeptide antibiotics. Microbiology (Reading, England), 152,2993. 

Miao, V., Coeffet-Le Gal, M.-F., Nguyen, K. et al. (2006) Genetic engineering in Streptomyces roseosporus to produce hybrid lipopeptide antibiotics. Chemistry Biology, 13, 269. 

Grunewald, J., Sieber, S.A., Mahlert, C. et al. (2004) Synthesis and derivatization of daptomycin a chemoenzy-matic route to acidic lipopeptide antibiotics. Journal of the American Chemical Society, 126, 17025. 

Kopp, F., Grunewald, J., Mahlert, C. and Marahiel, M.A. (2006) Chemoenzymatic design of acidic lipopeptide hybrids new insights into the structure-activity relationship of daptomycin and A54145FNR Biochemistry, 45, 10474. 

One of the most characteristic features of FRET is its sensitive dependency on the fluorophore distance. This is advantageously used to evaluate structures and conformational changes of peptides, glycopeptides, and proteins among other molecules [164-166], The conformational change of the lipopeptide antibiotic daptomycin from an inactive linear form to a biological active cyclic form... 

Surfactin Bacterial lipopeptide biosurfactant inhibits clot formation (387)... 

As well as fluorescence-based assays, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In a commercial BIA-core system [231] a hydrophobic SPR sensor with an alkane thiol surface was incubated with vesicles of defined size distribution generating a hybrid membrane by fusion of the lipid vesicles with the alkane thiol layer [232]. If the vesicles contain biotinylated lipopeptides their membrane anchoring can be analyzed by incubation with streptavidine. Accordingly, experiments with lipopeptides representing the C-terminal sequence of N-Ras show clear differences between single and double hydrophobic modified peptides in their ability to persist in the lipid layer [233]. 

New insights into the analysis of hydrophobically post-translational modified proteins could be achieved by the construction of lipidated proteins in a combination of bioorganic synthesis of activated lipopeptides and bacterial expression of the protein backbone (Fig. 19). The physico-chemical properties of such artificial lipoproteins differ substantially from those of the corresponding lipopeptides. The pronounced dominance of the hydrophilic protein moiety (e.g., for the Ras protein 181 amino acids) over a short lipopeptide with one or two hydrophobic modifications provides solubility up to 10 4 mol/1, while the biotinylated or fluorescence labeled lipopeptides exhibit low solubility in aqueous solutions and can be applied in the biophysical experiments only in vesicle integrated form or dissolved in organic solvent. 

Fig. 19. Lipopeptides with the aminoacid sequence of the Ras C-terminus and the natural or artificial lipid-modifications can be coupled with C-terminally truncated Ras via a maleimi-docaproyl linker. This electrophile reacts with free thiol groups (here, a C-terminal cysteine at the Ras moiety)...

Weigt, H.et al., The toll-loke receptor-2/6 agonist macrophage-activating lipopeptide-2 cooperates with IFN-gamma to reverse the Th2 skew in an in vitro allergy model, J. Immunol., 172, 6080, 2004. 

Heterodimerizes with TLR2 to recognize triacyl lipopeptides. 

For instance, in a synthesis of N-Ras lipopeptide 8, the choline ester in the palmitoylated tripeptide 5 was removed selectively and in high yield by means of the butyryl choline esterase (BChE). Efficient cou-... 

Hydrophobic and electrostatic properties of these lipopeptides show synergistic effects upon binding with membranes.1311 Due to the long stretch of basic amino acids the electrostatic interaction of the K-Ras4B peptide with negatively charged vesicles results in an approx. 103-fold increase in binding compared with a neutral membrane. 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

The insertion stability of several lipopeptides of the C-terminal sequence of N-Ras in such an artificial membrane is shown in Scheme 14. Here strepta-vidine is applied to indicate biotinylated lipopetides on the membrane surface. 

Eukaryotic cells utilize an efficient transport system that delivers macromolecules fast and secure to their destination. In the case of the small GTP binding proteins of the Ras family the modified C-terminus seems to be sufficient for addressing the polypeptide to its target membrane (in the case of Ras itself the plasma membrane). Lipopeptides with the C-terminal structure of N-Ras (either a pen-tamer with a C-terminal carboxymethylation and farnesylation or a heptapeptide with a palmitoyl thioester in addition) and a N-terminal 7-nitrobenz-2-oxa-l,3-diazolyl (NBD) fluorophore were microin-jected into NIH3T3 fibroblast cells and the distribution of the fluorophore was monitored by confocal laser fluorescence microscopy. Enrichment of the protein in the plasma membrane was efficient only for peptides with two hydrophobic modification sites, while the farnesylated but not palmitoylated peptide was distributed in the cytosol.1121... 

In a related experiment CV-1 fibroblasts were incubated with fluorescent N-Ras lipopeptides bearing a free palmitoylation site. These peptides cause staining of the CV-1 plasma membrane and efficient S-acylation even if the farnesyl group was replaced by a n-octyl group.1271 The association of the N-Ras lipopeptides with the plasma membrane... 

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