Location and dynamics of alamethicin in unilamellar vesicles and thylakoids as model systems. A spin label study.

Location and dynamics of the voltage-dependent pore-forming icosapeptide alamethicin have been studied using spin labels which were linked directly and via spacers to the C-terminus of the amphiphilic alpha-helix. Ion-transport activities of these derivatives were found to be very similar to those of natural alamethicin in green plant thylakoids chosen as a model system. The shape of the electron spin resonance spectra indicates segmental motion of the nitroxide rather than rotation of the whole peptide. A population of spins showing narrow lines in the presence of thylakoids or lipid vesicles is attributed to alamethicin in the aqueous solution. A second population shows rotational correlation times greater than 10(-9) s and is bound to the membranes, the C-termini residing in an environment with a polarity close to that of water. This population is inaccessible to the hydrophilic, charged line broadening agent chromium oxalate. Since spectral shapes and amplitudes of spectra are unchanged by additions of unlabelled peptide, it is concluded that the ESR detectable spins are bound to peptides essentially in the monomeric state. Alamethicin induced pore formation under flash illumination is demonstrated by measurement of kinetics of proton deposition in the thylakoid interior. When pores are opened by illuminating thylakoids and thus applying a membrane potential, mainly the bound population is affected by a process reversibly suppressing the signal, whereas only limited disappearance of label from the external medium is detected. Apparently, the potential causes a change in the conformation of the peptide which leads to a further immobilisation of the label, possibly due to a deeper insertion of the alpha-helices into the lipid membrane. However, evidence has been presented experimentally that there is no detectable change of potential prior to the opening of the pore.