A divalent-ion binding site on the 16-kDa proton channel from Nephrops norvegicus—revealed by EPR spectroscopy

As purified from the hepatopancreas of Nephrops norvegicus, the 16-kDa proton channel proteolipid is found to contain an endogenous divalent ion binding site that is occupied by Cu²⁺. The EPR spectrum has g-values and hyperfine splittings that are characteristic of type 2 Cu²⁺. The copper may be removed by extensive washing with EDTA. Titration with Ni²⁺ then induces spin-spin interactions with nitroxyl spin labels that are attached either to the unique Cys⁵⁴, or to fatty acids intercalated in the membrane. Paramagnetic relaxation enhancement by the fast-relaxing Ni²⁺ is used to characterise the binding and to estimate distances from the dipolar interactions. The Ni²⁺-binding site on the protein is situated around 14-18 Å from the spin label on Cys⁵⁴, and is at a similar distance from a lipid chain spin-labelled on the 5 C-atom, but is more remote from the C-9 and C-14 positions of the lipid chains.     

TOAC spin labels in the backbone of alamethicin: EPR studies in lipid membranes

Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe)(7,18,19) variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC(n) alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic (14)N-hyperfine couplings indicate that TOAC(8) and TOAC(16) are situated in the hydrophobic core of the membrane, whereas the TOAC(1) label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.     

Saturation transfer electron paramagnetic resonance spectroscopy of spin labeled tobacco mosaic virus protein.

The coat protein of Tobacco Mosaic Virus is covalently labeled with a maleimide spin label at the single SH-group of the protein. Saturation transfer electron paramagnetic resonance spectroscopy, a technique that is sensitive to very slow molecular motion with rotational correlation times τ_c in the range 10^?7 to 10^?3 sec, shows the dissociation of large oligomers of spin labeled protein with τ_c～10^?4 sec at pH 5.5 to smaller oligomers at higher pH.     

Lipid chain-length dependence for incorporation of alamethicin in membranes: electron paramagnetic resonance studies on TOAC-spin labeled analogs

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.