Alamethicin interaction with lipid membranes: a spectroscopic study on synthetic analogues.
Alamethicin (Alm) is one of the most extensively studied membrane-active antibiotic peptides, but several aspects of its mechanism of action are still under debate. In this study, synthetic analogues of natural Alm F50/5 (Alm-N), labeled with a 9H-fluoren-9-yl group at the N- (F-Alm) or C-terminus (Alm-F), were employed to investigate the position and orientation of this peptide in the membrane environment. Depth-dependent fluorescence quenching and polarized ATR-FT-IR experiments demonstrated that, in the absence of a transmembrane potential, Alm inserts its N-terminus into the membrane, while the C-terminus is exposed to the outer aqueous phase. We also found that the peptaibol populates different orientations with respect to the membrane normal. Furthermore, fluorescence resonance-energy transfer (FRET) indicated that no peptide translocation to the inner leaflet of lipid bilayers occurs. The mechanism of action of Alm is discussed on the basis of these findings. Two other Alm analogues, Alm-P and Alm-S, were exploited to investigate the role of specific Alm residues in terms of membrane-perturbing activity. Substitution of two or three Gln (E) residues (the only polar amino acids in the alamethicin sequence) by gamma-methyl glutamate (Glu(OMe)) residues induced marked variations in the aggregation and partition behaviors of the peptaibols, which, in turn, modulate their membrane activity. In particular, substitution of Gln(18) and Gln(19) caused a six-fold increase in membrane-perturbing activity, thus demonstrating that these residues are not essential for the stabilization of Alm pores.
Stella
Lorenzo
L
Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, Rome, Italy. stella@stc.uniroma2.it
Burattini
Marcello
M
Mazzuca
Claudia
C
Palleschi
Antonio
A
Venanzi
Mariano
M
Coin
Irene
I
Peggion
Cristina
C
Toniolo
Claudio
C
Pispisa
Basilio
B
D000408:Alamethicin / Q000737:chemistry*; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D008051:Lipid Bilayers; D008563:Membrane Lipids / Q000737:chemistry*; D008969:Molecular Sequence Data; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D008051:Lipid Bilayers; D008563:Membrane Lipids; D000408:Alamethicin