Specific PeptidePeptide Interactions and Lateral Crowding in Membranes
Specific Peptide-Peptide Interactions and Lateral Crowding in Membranes 19 Revealed by Solid State F NMR Stephan L. Grage 1, Sergii Afonin 1, Marco Ieronimo 2, Marina Berditsch 2, Parvesh Wadhwani 1, Pavel K. Mikhailiuk 2, 3, Igor V. Komarov 3, Anne S. Ulrich 1, 2 1 Institut für Biologische Grenzflächen, Forschungszentrum Karlsruhe, Hermann-von-Helmholtz-Platz 1, 76021 Karlsruhe, Germany, 2 Institut für Organische Chemie, Universität Karlsruhe, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany, 3 Organic Chemistry, Taras Shevchenko University, Kiev, 252033 Ukraine The Peptide PGLa Summary The activity of antimicrobial peptides relies on the interaction with the target membrane, where the interplay with the lipids, but also with proteinaceous components could be important. E. g. other antimicrobial peptides can modulate or synergistically enhance activity [1]. To gain insight in the influence of lateral crowding and specific peptide interactions on the antimicrobial mechanism, we analyzed the behaviour of PGLa in the presence of other peptides mimicking a proteinrich membrane. We found a pronounced change of the PGLa activity in the presence of Magainin-2, and confirmed the synergism of these two peptides [1]. The other peptides did not induce equivalent changes in the PGLa behaviour, indicating a specific interaction of PGLa and Magainin-2. Natural Different antimicrobial Model membrane activity in the presence membrane of proteins or peptides? K 19 G 1 A 8 K 12 K 15 K 5 S 4 V 16 We tested the effect of proteins on the activity of antimicrobial peptides using PGLa as an example. This a-helical peptide, derived from frog skin, forms a polar side with 4 lysines, opposed by an alanine-rich hydrophobic side. G 11 Different states of insertion into DMPC membranes were found recently for PGLa [2, 3]. At low protein: lipid ratios, PGLa lies flat on the bilayer surface (S-state). At high concentrations PGLa is surface aligned only at high temperatures. At temperatures just above the lipid phase transition, the peptide tilts into the membrane (T-state). Below the phase transition, PGLa inserts completely in a nearly upright position (I-state). The PGLa activity might be related to dimers formed in the T and I states. NMR to probe the peptide state H 2 N COOH I 9 L 18 G 7 A 14 19 F M 2 A 20 I 13 CF 3 A 6 A 3 A 10 A 17 L 21 1: 200 PGLa: lipid 1: 40 S 50°C S 35°C S S The insertion state of PGLa was monitored using 19 F-solid state NMR on oriented samples. To this aim we labelled PGLa with CF 3 bicyclopentylglycine, which links the CF 3 -label to the peptide backbone in a rigid way [4]. T I I -10 -30 -50 -70 -90 -110 -130 As a first step we evaluated which influence the peptides, used in this NMR study to mimick crowding, have on the antimicrobial activity of PGLa combined with Magainin-2 (MAG 2) leads to exhibition zones typical for a synergistic behaviour (see yellow arrows), whereas all other peptides do not change PGLa activity. In oriented samples, solid state NMR resonances become orientation dependent. Thus, position and splittings of the CF 3 -triplet signal reflect the orientation of PGLa. This way, all three states result in distinct 19 FNMR spectra. S T 15°C Results: Antimicrobial activity -150 Escherichia coli DH 5 a (gram-) Results: 19 F-NMR PGLa control Micrococcus luteus ATCC 4698 (gram+) 1: 200 S PGLa: lipid 1: 40 T S T I 55°C 50°C 45°C 40°C 35°C 30°C 25°C 20°C 15°C 1: 200 S T I 1: 200 PGLa: lipid 1: 40 S T I S I 1: 200 PGLa: lipid 1: 40 T S T I MAG 2: synergism MAP: helix-helix interactions? S T S S T I I 1: 200 PGLa: lipid 1: 40 T S T I I PGLa MAP GA GS PGLa MAP MAG 2 Samples were prepared with two PGLa concentrations: at low PGLa: DMPC (1: 200), where only the S-states occurred in the absence of a second peptide, and at high PGLa: DMPC (1: 40), where the inserted T and I states were found. The total peptide: lipid ratio was 1: 20. GS: surfacial crowding GA: transmembrane crowding GS MAG 2 d /ppm The insertion state of PGLa was followed by 19 F-NMR in the presence of a second peptide. We used gramicidin A (GA), gramicidin S (GS), a model-amphipatic peptide (MAP) and magainin-2 (MAG 2) to mimick crowding in different regions of the bilayer, and to probe specific interactions. GA Conclusion 0 The behaviour of PGLa changes only marginally in the presence of gramicidin A, gramicidin S or MAP. Magainin-2 on the other hand turns PGLa into the inserted I-state even at low PGLa concentrations. These results indicate that the presence of membrane proteins as such (“crowding”) has only little effect on the activity of antimicrobial peptides. Only when paired with a particular partner, such as magainin-2, the activity changes profoundly. The basis for the synergistic activity enhancement between PGLa and magainin-2 thus seems to be a specific interaction between these peptides. I Acknowlegdements We thank Erik Strandberg for providing his data analysis software. The DFG (CFN Karlsruhe) and Helmholtz Association are acknowledged for the financial support. Literature -10 -30 -50 -70 -90 -110 -130 -150 d /ppm -10 -30 -50 -70 -90 -110 -130 -150 d /ppm 0 -10 -30 -50 -70 -90 d /ppm -110 -130 -150 0 -10 -30 -50 -70 -90 d /ppm -110 -130 -150 [1] P. Tremouilhac et al (2006), J. Biol. Chem. 281, 32089 -94 [2] R. Glaser et al (2005), Biophysical Journal 88, 3392 -3397 [3] P. Tremouilhac (2006) BBA Biomembranes 1758, 1330 -1342 [4] P. K. Mikhailiuk et al (2006) Angewandte Chem. 45, 5659 -5661 Contact: stephan. grage@ibg. fzk. de
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