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.

FIGURE 1

Orientation of TOAC-labeled alamethicin in a lipid membrane. The principal molecular diffusion axis, R, is inclined at instantaneous angle γ to the membrane normal N. The nitroxide z axis is oriented at constant angle θ_z to R. The experimental order parameter, S_zz, of the nitroxide z axis is given by Eq. 5 where, for axial symmetry, 〈P₂(cosγ)〉 is the order parameter of the alamethicin diffusion axis.

FIGURE 2

Conventional EPR spectra (V₁-display) of [Glu(OMe)^7,18,19] alamethicin analogs with TOAC substituted for: residue 1, TOAC¹; residue 8, TOAC⁸; or residue 16, TOAC¹⁶ in phosphatidylcholine bilayers of chain lengths C₁₀–C₁₈, as indicated, at 75°C. Total scan width = 100 G.

FIGURE 3

Temperature dependences of the outer hyperfine splittings, 2A_max, for [Glu(OMe)^7,18,19] alamethicin TOAC¹ (squares), TOAC⁸ (circles), and TOAC¹⁶ (triangles) analogs in phosphatidylcholine bilayers of chain lengths C₁₀–C₁₈, as indicated.

FIGURE 4

Temperature dependences of the effective isotropic hyperfine couplings, for [Glu(OMe)^7,18,19] alamethicin TOAC¹ (squares), TOAC⁸ (circles), and TOAC¹⁶ (triangles) analogs in phosphatidylcholine bilayers of chain lengths C₁₀–C₁₈, as indicated.

FIGURE 5

Left-hand panel: chain-length dependence (N) of the effective isotropic hyperfine couplings, a_o, for [Glu(OMe)^7,18,19] alamethicin TOAC¹ (squares), TOAC⁸ (circles), and TOAC¹⁶ (triangles) analogs in fluid diC_NPtdCho bilayers. Right-hand panel: positional dependence (n) of the isotropic hyperfine couplings for nitroxides at position C-n in the sn-2 chain of n-PCSL phosphatidylcholine spin probes in diC₁₄PtdCho (circles) or diC₁₆PtdCho (squares) fluid bilayers (20). Values of a_o are given relative to those in methanol: Δa_o = a_o(PtdCho) − a_o(MeOH). Horizontal dotted lines are the values of Δa_o in MeOH and in EtOH, as indicated.

FIGURE 6

Chain-length dependences (N) of the effective order parameters, S_zz, for [Glu(OMe)^7,18,19] alamethicin TOAC¹ (squares), TOAC⁸ (circles), and TOAC¹⁶ (triangles) analogs in diC_NPtdCho bilayers at 75°C (solid symbols) and 80°C (open symbols).

FIGURE 7

Solid lines: chain-length dependence (N) of the order parameters, 〈P₂(cosγ)〉, for the alamethicin diffusion axis in diC_NPtdCho bilayers at 75°C (solid squares) and 80°C (solid circles). Dotted lines: orientation, P₂(cosθ_z), of the nitroxide z axis for TOAC¹ (open squares), TOAC⁸ (open circles), and TOAC¹⁶ (open triangles) in [Glu(OMe)^7,18,19] alamethicin analogs. Values are determined from nonlinear least squares fitting of Eq. 5 to the chain-length dependence of S_zz measured at 75°C and 80°C, with constant θ_z for each TOAC position (see text and Fig. 1).

FIGURE 8

Saturation transfer EPR spectra (V₂′-display) of [Glu(OMe)^7,18,19] alamethicin analogs with TOAC substituted for: residue 1, TOAC¹; residue 8, TOAC⁸; and residue 16, TOAC¹⁶ in phosphatidylcholine bilayers of chain lengths C₁₀–C₁₈ at the temperatures indicated. Total scan width = 160 G.

FIGURE 9

Temperature dependences of the integrated intensity, I_ST, from the ST-EPR spectra of the TOAC⁸ [Glu(OMe)^7,18,19] alamethicin analog in diC_NPtdCho bilayers with N = 12 (squares), 14 (circles), 16 (triangles), and 18 (diamonds). Data points for N = 10 superimpose on those for N = 12.

FIGURE 10

Chain-length dependences of the thickness of diC_NPtdCho bilayers for N = 12, 14 (34), and 16 (35) at 50°C. Solid squares: thickness of the hydrocarbon core, d_C; open squares: anhydrous bilayer thickness, d_b; open circles: steric thickness of the bilayer, d_st = d_C + 1.8 nm. Measurements of d_C and d_b for N = 12 and 14 at 30°C are corrected to 50°C by using an expansion coefficient α_d = (1/d)(∂d/∂T) = −0.0032K⁻¹ (34). Sloping lines are linear regressions. The approximate position of [Glu(OMe)^7,18,19] alamethicin in diC₁₄PtdCho and diC₁₆PtdCho bilayers, consistent with Fig. 5, is indicated by the heavy vertical line and horizontal dotted lines (C^α-atoms), which are located at ∼ +1.45, +0.4, and −0.8 nm for TOAC¹, TOAC⁸, and TOAC¹⁶, respectively.

FIGURE 11

Predicted dependence of the order parameter of the long axis of alamethicin on chain length of host diC_NPtdCho bilayers according to Eq. 6 (solid lines) or Eq. 7 (dashed lines) and Eq. 8 for hydrophobic matching. Dimensions of the lipid hydrocarbon core, d_C, are taken from Fig. 10, and the hydrophobic span of alamethicin is d_p = 2.8 nm or 3.4 nm, as indicated. Squares (circles) are order parameter measurements at 75°C (85°C); dotted lines are nonlinear fits of Eqs. 7 and 8 with and Δd_C/d_p = 0.0147 ± 0.0006 (0.0159 ± 0.0005).