Structure of (KIAGKIA)3 Aggregates in Phospholipid Bilayers by Solid-State NMR

Abstract

The interchain (13)C-(19)F dipolar coupling measured in a rotational-echo double-resonance (REDOR) experiment performed on mixtures of differently labeled KIAGKIA-KIAGKIA-KIAGKIA (K3) peptides (one specifically (13)C labeled, and the other specifically (19)F labeled) in multilamellar vesicles of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (1:1) shows that K3 forms close-packed clusters, primarily dimers, in bilayers at a lipid/peptide molar ratio (L/P) of 20. Dipolar coupling to additional peptides is weaker than that within the dimers, consistent with aggregates of monomers and dimers. Analysis of the sideband dephasing rates indicates a preferred orientation between the peptide chains of the dimers. The combination of the distance and orientation information from REDOR is consistent with a parallel (N-N) dimer structure in which two K3 helices intersect at a cross-angle of approximately 20 degrees. Static (19)F NMR experiments performed on K3 in oriented lipid bilayers show that between L/P = 200 and L/P = 20, K3 chains change their absolute orientation with respect to the membrane normal. This result suggests that the K3 dimers detected by REDOR at L/P = 20 are not on the surface of the bilayer but are in a membrane pore.

Antimicrobial peptides: New candidates in the fight against bacterial infections

“…Recently, a solid-state NMR investigation of K3, 108 a synthetic analogue of PGLa, has provided a direct structural evidence of peptide dimerization in PC/PG bilayers. 141 The combination of distance and orientation information obtained from rotationalecho double resonance (REDOR) 142 experiments on mixtures of differently labeled K3 peptides have indicated a parallel dimer structure in which K3 helices intersect at a cross-angle of approximately 208.…”

“…145 While the barrel-stave mechanism seems to hold for noncell-selective lytic peptides only, the carpet mechanism appears to be a more general permeabilization mechanism that is responsible for the lytic action of peptides in negatively charged lipid bilayers such as the outer leaflet of the bacterial cell membrane. 11 The list includes cecropins, 146 141 melittin, 156 and the -sheet protegrin. 176 We should also note that in the case of melittin, there have been other earlier and more recent reports that indicate the peptide may form barrel-stave-type pores in zwitterionic membranes 101,102,177,178 and act via a detergent-like permeabilization mechanism in anionic lipids.…”

“…The interactions between transmembrane helices in ion channels tend to be highly specific 180,181 : hydrogen bonds, possible salt bridges, interactions between helix dipoles, and van der Waals interactions (this also includes packing effects, i.e., differences between helix-helix vs. helix-lipid chain packing) lead to close packing of the helices, overcoming the unfavorable entropic effect that arises upon the association of initially separated polypeptide chains. The recruitment of subsequent helices in a stepwise manner is indicated, for instance, by the discrete conductance levels observed for alamethicin pores, 182 Unlike the large peptide aggregates in the barrelstave mechanism, self-association to a smaller extent has been reported for magainins 139,141 and some of the dermaseptins, 147,148 peptides thought to act in lipid bilayers via the toroidal mechanism and carpetmechanism, respectively.…”

NMR methods for studying membrane‐active antimicrobial peptides

“…The strong 13 C-31 P dipolar coupling showed that the peptide must lie flat on the membrane surface, in order for all carbons to be close to a phospholipid head group. More recently, a combination of different 13 C/ 15 N/ 19 F/ 31 P REDOR experiments on the antimicrobial peptide K3 have demonstrated that it can self-assemble into head-to-head dimers at high concentrations in the membrane (99,100). Finally, a different type of experiment using slow sample spinning, 2D SSRS-MASE, has revealed the relative orientation of two CO CSA tensors on consecutive amino acids in melittin (88).…”

“…When this approach was applied to PGLa, it revealed different tilt angles of the peptide in the membrane, depending on the peptide concentration and lipid composition (74,116). The re-alignment was interpreted in terms of dimerization, in analogy to the results on the related K3 peptide that has been extensively characterized from REDOR distances, including 19 F-13 C constraints (99,100). In a different approach, 19 F NMR can be used to probe the depth of membrane immersion by O 2 quenching.…”