Nisin is a 34 residue long peptide belonging to the group A lantibiotics with antimicrobial activity against Gram-positive bacteria. The antimicrobial activity is based on pore formation in the cytoplasmic membrane of target organisms. The mechanism which leads to pore formation remains to be clarified. We studied the orientation of nisin via site-directed tryptophan fluorescence spectroscopy. Therefore, we engineered three nisin Z variants with unique tryptophan residues at positions 1, 17, and 32, respectively. The activity of the tryptophan mutants against Gram-positive bacteria and in model membrane systems composed of DOPC or DOPG was established to be similar to that of wild type nisin Z. The tryptophan fluorescence emission maximum showed an increasing blue-shift upon interaction with vesicles containing increased amounts of DOPG, with the largest effect for the 1W peptide. Studies with the aqueous quencher acrylamide showed that all tryptophans became inaccessible from the aqueous phase in the presence of negatively charged lipids in the vesicles. From these results it is concluded that anionic lipids mediate insertion of the tryptophan residues in at least three positions of the molecule into the lipid bilayer. The depth of insertion of the tryptophan residues was determined via quenching of the tryptophan fluorescence by spin-labeled lipids. The results showed that the depth of insertion was dependent on the amount of negatively charged lipids. In membranes containing 50% DOPG, the distances from the bilayer center were determined to be 15.7, 15.0, and 18.4 A for the tryptophan at position 1, 17, and 32, respectively. In membranes containing 90% DOPG, these distances were calculated to be 10.8, 11.5, and 13.1 A, respectively. These results suggest an overall parallel average orientation of nisin in the membrane, with respect to the membrane surface, with the N-terminus more deeply inserted than the C-terminus. These data were used to model the orientation of nisin in the membrane.

Recently we observed in an in vitro system that newly synthesized KcsA assembles e⁄ciently into a tetramer in lipid vesicles [van Dalen et al. (2002) FEBS Lett. 511, 51^58]. Here we used this system to get insight into the importance of the lipid composition for KcsA membrane association and tetramerization and we compared this to the lipid dependency of the thermo-stability of the KcsA tetramer. It was found that a large amount of phosphatidylethanolamine ( s 40 mol%) and a lower amount of phosphatidylglycerol (V V20^30 mol%) were optimal for e⁄cient KcsA membrane association and tetramerization. Strikingly, vesicles of the abundant and commonly used membrane lipid phosphatidylcholine did not support assembly, further demonstrating the importance of membrane lipid composition for KcsA assembly. The in vitro assembled KcsA tetramer showed similar thermo-stability in biological and pure lipid membranes, demonstrating that both tetramers are alike. In addition, we show that solubilization of the membrane with detergent reduces the thermo-stability of the tetramer. The highest KcsA tetramer stability was observed in intact bilayers in the presence of anionic lipids. ß

The mechanism by which phospholipids are transported across biogenic membranes, such as the bacterial cytoplasmic membrane, is unknown. We hypothesized that this process is mediated by the presence of the membrane-spanning segments of inner membrane proteins, rather than by dedicated flippases. In support of the hypothesis, it was demonstrated that transmembrane ␣-helical peptides, mimicking the membrane-spanning segments, mediate flop of 2-6-(7-nitro-2,1,3-benzoxadiazol-4-yl) aminocaproyl (

In this study we have used electrospray ionization mass spectrometry (ESI-MS) to investigate interactions between the bacterial K + channel KcsA and membrane phospholipids. KcsA was reconstituted into lipid vesicles of variable lipid composition. These vesicles were directly analyzed by ESI-MS or mixed with tri£uoroethanol (TFE) before analysis. In the resulting mass spectra, non-covalent complexes of KcsA and phospholipids were observed with an interesting lipid speci¢city. The anionic phosphatidylglycerol (PG), and, to a lesser extent, the zwitterionic phosphatidylethanolamine (PE), which both are abundant bacterial lipids, were found to preferentially associate with KcsA as compared to the zwitterionic phosphatidylcholine (PC). These preferred interactions may re£ect the di¡erences in a⁄nity of these phospholipids for KcsA in the membrane.