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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.
Biophysical Journal 05/2005; 88(5):3392-7. · 3.65 Impact Factor
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ABSTRACT: A highly sensitive solid state (19)F-NMR strategy is described to determine the orientation and dynamics of membrane-associated peptides from specific fluorine labels. Several analogues of the antimicrobial peptide PGLa were synthesized with the non-natural amino acid 4-trifluoromethyl-phenylglycine (CF(3)-Phg) at different positions throughout the alpha-helical peptide chain. A simple 1-pulse (19)F experiment allows the simultaneous measurement of both the anisotropic chemical shift and the homonuclear dipolar coupling within the rotating CF(3)-group in a macroscopically oriented membrane sample. The value and sign of the dipolar splitting determines the tilt of the CF(3)-rotational axis, which is rigidly attached to the peptide backbone, with respect to the external magnetic field direction. Using four CF(3)-labeled peptide analogues (with L-CF(3)-Phg at Ile9, Ala10, Ile13, and Ala14) we confirmed that PGLa is aligned at the surface of lipid membranes with its helix axis perpendicular to the bilayer normal at a peptide:lipid ratio of 1:200. We also determined the azimuthal rotation angle of the helix, which agrees well with the orientation expected from its amphiphilic character. Peptide analogues with a D-CF(3)-Phg label resulting from racemization of the amino acid during synthesis were separately collected by HPLC. Their spectra provide additional information about the PGLa structure and orientation but allow only to discriminate qualitatively between multiple solutions. The structural and functional characterization of the individual CF(3)-labeled peptides by circular dichroism and antimicrobial assays showed only small effects for our four substitutions on the hydrophobic face of the helix, but a significant disturbance was observed in a fifth analogue where Ala8 on the hydrophilic face had been replaced. Even though the hydrophobic CF(3)-Phg side chain cannot be utilized in all positions, it allows highly sensitive NMR measurements over a wide range of experimental conditions and dynamic regimes of the peptide.
Journal of Magnetic Resonance 06/2004; 168(1):153-63. · 2.14 Impact Factor
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ABSTRACT: A highly sensitive solid state 19F-NMR strategy is described to determine the orientation and dynamics of membrane-associated peptides from specific fluorine labels. Several analogues of the antimicrobial peptide PGLa were synthesized with the non-natural amino acid 4-trifluoromethyl-phenylglycine (CF3-Phg) at different positions throughout the α-helical peptide chain. A simple 1-pulse 19F experiment allows the simultaneous measurement of both the anisotropic chemical shift and the homonuclear dipolar coupling within the rotating CF3-group in a macroscopically oriented membrane sample. The value and sign of the dipolar splitting determines the tilt of the CF3-rotational axis, which is rigidly attached to the peptide backbone, with respect to the external magnetic field direction. Using four CF3-labeled peptide analogues (with l-CF3-Phg at Ile9, Ala10, Ile13, and Ala14) we confirmed that PGLa is aligned at the surface of lipid membranes with its helix axis perpendicular to the bilayer normal at a peptide:lipid ratio of 1:200. We also determined the azimuthal rotation angle of the helix, which agrees well with the orientation expected from its amphiphilic character. Peptide analogues with a d-CF3-Phg label resulting from racemization of the amino acid during synthesis were separately collected by HPLC. Their spectra provide additional information about the PGLa structure and orientation but allow only to discriminate qualitatively between multiple solutions. The structural and functional characterization of the individual CF3-labeled peptides by circular dichroism and antimicrobial assays showed only small effects for our four substitutions on the hydrophobic face of the helix, but a significant disturbance was observed in a fifth analogue where Ala8 on the hydrophilic face had been replaced. Even though the hydrophobic CF3-Phg side chain cannot be utilized in all positions, it allows highly sensitive NMR measurements over a wide range of experimental conditions and dynamic regimes of the peptide.
Journal of Magnetic Resonance.
