Conformation of spin-labeled melittin at membrane surfaces investigated by pulse saturation recovery and continuous wave power saturation electron paramagnetic resonance.

Melittin spin-labeled specifically with a nitroxide at positions 7, 21, 23, or the amino terminus was bound to phospholipid membranes, and the exposure of the spin label to the aqueous phase was investigated by measurement of Heisenberg exchange with chromium oxalate in the solution. The exchange frequency was determined by saturation recovery electron paramagnetic resonance (EPR) using a loop-gap resonator. This method allows use of very low concentrations (less than 1 mM) of chromium oxalate compared with conventional measurements of EPR line broadening (typically 50 mM), thus avoiding problems associated with high metal ion concentration. Differences in exchange frequency between the various positions were also estimated by continuous wave power saturation methods. In either approach, the spin label at lysine 7 was found to be the most exposed to chromium oxalate whereas that at lysine 23 was found to be the least exposed. This is consistent with a model for the membrane bound peptide in which an amphiphilic helix lies with its axis parallel to the bilayer surface and the hydrophobic moment points toward the bilayer interior

Multifrequency ESR with Fourier Analysis of Cu"(His), (His = Histidine). 2. Mobile Phase'

Mobile-phase Cu(His)2 complexes in water at room temperature in the presence of excess histidine are characterized by ESR methods. In order to identify the nitrogen donor atom, histidine, for which I4N in the imidazole ring is substituted with I5N, was used. Spectra in the presence of excess histidine have been analyzed by simulation of second-derivative spectra and by Fourier and reverse Fourier transforms of the spectra. It is concluded that the spectra arise from a superposition of an 80% contribution from complexes formed with two histidines in a histamine-like configuration and a 20% contribution from complexes with two histidines: one glycine-like and one histamine-like. It is suggested that Fourier and reverse Fourier transforms are particularly effective methods to analyze the nitrogen superhyperfine patterns in spectra from copper-histidine complexes