Concentration-Dependent Realignment of the Antimicrobial Peptide PGLa in Lipid Membranes Observed by Solid-State 19F-NMR

Abstract

The membrane-disruptive antimicrobial peptide PGLa is found to change its orientation in a dimyristoyl-phosphatidylcholine bilayer when its concentration is increased to biologically active levels. The alignment of the alpha-helix was determined by highly sensitive solid-state NMR measurements of (19)F dipolar couplings on CF(3)-labeled side chains, and supported by a nonperturbing (15)N label. At a low peptide/lipid ratio of 1:200 the amphiphilic peptide resides on the membrane surface in the so-called S-state, as expected. However, at high peptide concentration (>/=1:50 molar ratio) the helix axis changes its tilt angle from approximately 90 degrees to approximately 120 degrees , with the C-terminus pointing toward the bilayer interior. This tilted "T-state" represents a novel feature of antimicrobial peptides, which is distinct from a membrane-inserted I-state. At intermediate concentration, PGLa is in exchange between the S- and T-state in the timescale of the NMR experiment. In both states the peptide molecules undergo fast rotation around the membrane normal in liquid crystalline bilayers; hence, large peptide aggregates do not form. Very likely the obliquely tilted T-state represents an antiparallel dimer of PGLa that is formed in the membrane at increasing concentration.

Synergistic Transmembrane Alignment of the Antimicrobial Heterodimer PGLa/Magainin

“…Choice of Peptide-Lipid System-For PGLa in DMPC bilayers, we have found previously that the peptide binds in a monomeric S-state at P/L Յ 1:200, whereas at P/L Ն 1:50, a homodimeric T-state is characterized in oriented NMR samples (17,21). In a negatively charged lipid mixture of DMPC/ DMPG (3:1), the same realignment of PGLa was observed as in zwitterionic DMPC, although the T-state was reached at a somewhat lower peptide concentration.…”

“…The structure analysis from the 2 H-NMR data was performed as previously described for PGLa (17,18,20,21,28). This method is described in depth in several recent reviews (3,26,36).…”

“…In determining whether PGLa has a preference for forming heterodimers with MAG rather than homodimers, we expected to see structural differences compared with its usual behavior, which we had characterized in detail previously (17)(18)(19)(20)(21). The amino acid sequences (see Table 1) suggest that both peptides form amphiphilic ␣-helices, with charged and polar side chains on one face and hydrophobic residues on the other (see helical wheels in Fig.…”

“…We therefore used an ideal poly(alanine) ␣-helix as a model structure for PGLa, and this model was fitted to the experimental 2 H-NMR data of the peptide. In a grid search for the best-fit structure, the theoretical quadrupole splittings were systematically calculated for all combinations of , , and S mol (17,18,21). The parameters and were changed in steps of 1°a nd S mol in steps of 0.01 to find the lowest root mean square deviation with regard to the experimental data.…”

Effect of Cholesterol on the Phase Change of Lipid Membranes by Antimicrobial Peptides

“…A micelle 30 or a vesicle 31 has been used to simulate a biomembrane and, recently, a bicelle 32 with a flat surface is widely used. Spectroscopic methods such as circular dichroism, 33 neutron diffraction, 25 X-ray diffraction, 34 and nuclear magnetic resonance spectroscopy 35,36 have been used to analyze the action modes and the binding structure of AMPs. For example, the binding pattern of Psd1 and partition to lipid membranes containing ergosterol or cholesterol were studied using fluorescence spectroscopy.…”