Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels

Gläser, Roland; Sachse, Carsten; Dürr, U.; Wadhwani, Parvesh; Ulrich, Anne S.

doi:10.1016/j.jmr.2004.02.008

Cited by 109 publications

(220 citation statements)

References 22 publications

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“…1). For PGLa, the ␣-helical conformation is confirmed by circular dichroism in the presence of lipid vesicles (15,20,22) and by 1 H-NMR in detergent micelles (23). MAG is also known to form an ␣-helix when bound to membranes (24).…”

mentioning

confidence: 89%

“…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.…”

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confidence: 97%

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Synergistic Transmembrane Alignment of the Antimicrobial Heterodimer PGLa/Magainin

Tremouilhac

Strandberg

Wadhwani

et al. 2006

Journal of Biological Chemistry

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102

128

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The antimicrobial activity of amphipathic ␣-helical peptides is usually attributed to the formation of pores in bacterial membranes, but direct structural information about such a membranebound state is sparse. Solid state 2 H-NMR has previously shown that the antimicrobial peptide PGLa undergoes a concentrationdependent realignment from a surface-bound S-state to a tilted T-state. The corresponding change in helix tilt angle from 98 to 125°was interpreted as the formation of PGLa/magainin heterodimers residing on the bilayer surface. Under no conditions so far, has an upright membrane-inserted I-state been observed in which a transmembrane helix alignment would be expected. Here, we have demonstrated that PGLa is able to assume such an I-state in a 1:1 mixture with magainin 2 at a peptide-to-lipid ratio as low as 1:100 in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol model membranes. This 2 H-NMR analysis is based on seven orientational constraints from Ala-3,3,3-d 3 substituted in a non-perturbing manner for four native Ala residues as well as two Ile and one Gly. The observed helix tilt of 158°is rationalized by the formation of heterodimers. This structurally synergistic effect between the two related peptides from the skin of Xenopus laevis correlates very well with their known functional synergistic mode of action. To our knowledge, this example of PGLa is the first case where an ␣-helical antimicrobial peptide is directly shown to assume a transmembrane state that is compatible with the postulated toroidal wormhole pore structure.

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confidence: 89%

mentioning

confidence: 97%

Synergistic Transmembrane Alignment of the Antimicrobial Heterodimer PGLa/Magainin

Tremouilhac

Strandberg

Wadhwani

et al. 2006

Journal of Biological Chemistry

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102

128

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“…Other periodicities are indicative of different helix types. Note that, in practice it is often only possible to obtain the absolute value of a quadrupolar or dipolar splitting (74). Furthermore, from an anisotropic "wave" pattern it is possible to determine the tilt angle and azimuthal rotation of the helix in the membrane (65,75).…”

Section: Simple Secondary Structuresmentioning

confidence: 99%

“…In order to obtain orientational information, it is, therefore, more promising to analyze the anisotropic intramolecular dipolar coupling within the CF 3 -Phg group. 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).…”

Section: F Nmr On Peptidesmentioning

confidence: 99%

NMR methods for studying membrane‐active antimicrobial peptides

Strandberg

Ulrich

2004

Concepts Magnetic Resonance

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NMR is a versatile tool for studying interactions between antimicrobial peptides and lipid membranes. Different approaches using both liquid state and solid state NMR are outlined here, with an emphasis on solid state NMR methods, to study the structures of antimicrobial peptides in lipid bilayers as well as the effect of these peptides on model membranes. Different NMR techniques for observing both peptides and lipids are explained, including

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“…Gramicidin S (GS) and PGLa are characterized by a ␤-stranded and an ␣-helical conformation, respectively, and their behavior in model lipid membranes has been thoroughly investigated by our group using solid-state nuclear magnetic resonance (NMR) (1)(2)(3)17). To extend the current, quasiatomistic picture of these AMPs in terms of their morphological impact on the native membranes of living bacteria, we have now used EM to examine the bacterial morphology after treatment with GS and PGLa.…”

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confidence: 99%

Damage of the Bacterial Cell Envelope by Antimicrobial Peptides Gramicidin S and PGLa as Revealed by Transmission and Scanning Electron Microscopy

Hartmann¹,

Berditsch

Hawecker

et al. 2010

Antimicrob Agents Chemother

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Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the ultrastructural changes in bacteria induced by antimicrobial peptides (AMPs). Both the ␤-stranded gramicidin S and the ␣-helical peptidyl-glycylleucine-carboxyamide (PGLa) are cationic amphiphilic AMPs known to interact with bacterial membranes. One representative Gram-negative strain, Escherichia coli ATCC 25922, and one representative Gram-positive strain, Staphylococcus aureus ATCC 25923, were exposed to the AMPs at sub-MICs and supra-MICs in salt-free medium. SEM revealed a shortening and swelling of the E. coli cells, and multiple blisters and bubbles formed on their surface. The S. aureus cells seemed to burst upon AMP exposure, showing open holes and deep craters in their envelope. TEM revealed the formation of intracellular membranous structures in both strains, which is attributed to a lateral expansion of the lipid membrane upon peptide insertion. Also, some morphological alterations in the DNA region were detected for S. aureus. After E. coli was incubated with AMPs in medium with low ionic strength, the cells appeared highly turgid compared to untreated controls. This observation suggests that the AMPs enhance osmosis through the inner membrane, before they eventually cause excessive leakage of the cellular contents. The adverse effect on the osmoregulatory capacity of the bacteria is attributed to the membrane-permeabilizing action of the amphiphilic peptides, even at low (sub-MIC) AMP concentrations. Altogether, the results demonstrate that both TEM and SEM, as well as appropriate sample preparation protocols, are needed to obtain detailed mechanistic insights into peptide function.

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Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels

Cited by 109 publications

References 22 publications

Synergistic Transmembrane Alignment of the Antimicrobial Heterodimer PGLa/Magainin

Synergistic Transmembrane Alignment of the Antimicrobial Heterodimer PGLa/Magainin

NMR methods for studying membrane‐active antimicrobial peptides

Damage of the Bacterial Cell Envelope by Antimicrobial Peptides Gramicidin S and PGLa as Revealed by Transmission and Scanning Electron Microscopy

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