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Conformation of Alamethicin in Oriented Phospholipid Bilayers Determined by 15N Solid-State Nuclear Magnetic Resonance

2001; Elsevier BV; Volume: 81; Issue: 3 Linguagem: Inglês

10.1016/s0006-3495(01)75822-5

1542-0086

Mads Bak, Robert P. Bywater, M. Hohwy, J. Thomsen, Kim Adelhorst, Hans J. Jakobsen, Ole W. Sørensen, Niels Chr. Nielsen,

Protein Structure and Dynamics

Abstract The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using 15 N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently 15 N-labeled variants of alamethicin and an analog with three of the α -amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro 14 residue. From the anisotropic 15 N chemical shifts and 1 H– 15 N dipolar couplings determined for alamethicin with 15 N-labeling on the Ala 6 , Val 9 , and Val 15 residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear α -helical structure spanning the membrane with the molecular axis tilted by 10–20° relative to the bilayer normal. In particular, we find compatibility with a straight α -helix tilted by 17° and a slightly kinked molecular dynamics structure tilted by 11° relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325–330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.