Lipid Chain-Length Dependence for Incorporation of Alamethicin in Membranes: Electron Paramagnetic Resonance Studies on TOAC-Spin Labeled Analogs

Abstract

Alamethicin is a 19-residue hydrophobic peptide, which is extended by a C-terminal phenylalaninol but lacks residues that might anchor the ends of the peptide at the lipid-water interface. Voltage-dependent ion channels formed by alamethicin depend strongly in their characteristics on chain length of the host lipid membranes. EPR spectroscopy is used to investigate the dependence on lipid chain length of the incorporation of spin-labeled alamethicin in phosphatidylcholine bilayer membranes. The spin-label amino acid TOAC is substituted at residue positions n = 1, 8, or 16 in the sequence of alamethicin F50/5 [TOAC(n), Glu(OMe)(7,18,19)]. Polarity-dependent isotropic hyperfine couplings of the three TOAC derivatives indicate that alamethicin assumes approximately the same location, relative to the membrane midplane, in fluid diC(N)PtdCho bilayers with chain lengths ranging from N = 10-18. Residue TOAC(8) is situated closest to the bilayer midplane, whereas TOAC(16) is located farther from the midplane in the hydrophobic core of the opposing lipid leaflet, and TOAC(1) remains in the lipid polar headgroup region. Orientational order parameters indicate that the tilt of alamethicin relative to the membrane normal is relatively small, even at high temperatures in the fluid phase, and increases rather slowly with decreasing chain length (from 13 degrees to 23 degrees for N = 18 and 10, respectively, at 75 degrees C). This is insufficient for alamethicin to achieve hydrophobic matching. Alamethicin differs in its mode of incorporation from other helical peptides for which transmembrane orientation has been determined as a function of lipid chain length.

Polarity dependence of EPR parameters for TOAC and MTSSL spin labels: Correlation with DOXYL spin labels for membrane studies

“…Practical considerations for the measurement of a N o from anisotropic spin-label EPR spectra in biological systems are given in Refs. [12,13,17,18]. For aprotic environments, the necessary relations are established by a common effective dielectric constant.…”

“…The nitroxide ring of TOAC is incorporated rigidly in the peptide backbone and therefore is optimally situated to determine the location of the various secondary structural elements of the peptide. Sensitivity of the isotropic hyperfine couplings to membrane location has been demonstrated for TOAC-labelled trichogin GA IV [16] and alamethicin F50/5 [17,18] antimicrobial peptaibols. Positional specificity in nitroxide-labelling of proteins is now obtained routinely in site-directed spin-labelling [19][20][21].…”

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR

“…Continuous-wave electron paramagnetic resonance (EPR) studies at room temperature suggest that the majority of the membrane-bound ALM molecules are probably in the monomeric transmembrane oriented state, and upon a reduction in temperature they begin to self-assemble below the lipid-phase transition temperature (25). Ionic conductances observed at room temperature probably arise from transient and/or voltage-induced molecular associations (26). X-ray and neutron diffraction techniques have provided information about the structure factor of the pore fluid and the influence of peptides on the phase and thickness of the membrane (27)(28)(29)(30)(31)(32).…”

“…The 19 F labeling of [Glu(OMe) 7,18,19 ] ALM F50/5 was achieved by synthesis of the analog ALM9, wherein the hydrophobic Val 9 , situated in the middle of the sequence, was replaced by the hydrophobic and lipophobic Dab(Tfa) 9 residue (26,(40)(41)(42)(43). Here, we conducted a 19 F MAS solid-state NMR investigation of ALM9 to study its aggregation in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid membranes using the CODEX approach (38).…”

Reaction fields and solvent dependence of the EPR parameters of nitroxides: The microenvironment of spin labels

“…The polarisability of the nitroxide is included in every case, and is found to have a particularly significant effect for the Wertheim model. An analysis such as this is important not only for comparison of spin-labelled biological environments with solutions of defined polarity, but also for transfer of polarity dependences between different nitroxide spin labels [28,29]. The latter is particularly significant for transmembrane polarity profiles that are established mostly by oxazolidine-nitroxide spin-labelled lipids [30][31][32], whereas integral membrane peptides and proteins are mostly spin-labelled with piperidine-, pyrrolidine-or pyrroline-nitroxide derivatives (e.g., Refs.…”