Aggregation of an α-Helical Transmembrane Peptide in Lipid Phases, Studied by Time-Resolved Fluorescence Spectroscopy

The aggregation of octadecyl rhodamine 101 (Rh101C18) and a Rh101-labeled transmembrane peptide (Rh101WALP16) solubilized in lipid phases was studied. Different lipid phases in excess of water were formed with either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or 1,2-dierucoyl-sn-glycero-3-phosphochline (DErPC). Rh101 was covalently attached with its carboxyl group to the C-terminal ethanolamine. Time-resolved polarized fluorescence spectroscopy was used to determine the fluorescence relaxation and anisotropy at molar ratios ranging from 1 Rh101derivative per 5000 lipids (1:5000) to 1 Rh101-derivative per 50 lipids (1:50). At concentrations of the Rh101derivatives exceeding about 1 mol %, the fluorescence intensity is quenched, and the fluorescence lifetime is significantly decreased. This is compatible with electronic energy transfer to ground-state dimers of Rh101groups. The time-resolved fluorescence decays were analyzed by analytical models of donor-acceptor electronic energy transfer. The models account for energy transfer between Rh101 monomers (donors) and Rh101dimers (acceptors), spatially distributed in one and two dimensions. For the amphiphilic Rh101C18 molecules solubilized in DMPC, DOPC, and DErPC, the fluorescence relaxation is very well described as energy transfer in lipid vesicles between donors and acceptors, distributed on the inner and outer surfaces of the bilayer. For statistical reasons, pairs of molecules in contact are more likely to appear at high concentrations. Hereafter these are referred to as statistical dimers. It is found that the dimer concentration of Rh101C 18 in the bilayer of the various lipids, is slightly above that calculated for statistical dimers. In contrast, the dimerization of Rh101WALP16 is five- to 10-fold increased, as compared to the expected concentration of statistical dimers. This suggests that the transmembrane peptide has an inherent affinity for aggregation in lipid bilayers formed by DMPC, DOPC, and DErPC. The fluorescence relaxation of Rh101WALP16 in DMPC and DOPC is very well described as donor-acceptor energy transfer across the lipid bilayer of vesicles. However, the fluorescence relaxation of Rh101WALP16 in DErPC is not compatible with energy transfer in lipid bilayers. Instead, a better description is achieved by assuming that the Rh101WALP16 molecules are aggregated along parallel lines. Such a spatial distribution is compatible with recent studies showing a reversed hexagonal phase (HII) that appears at high WALP16 concentrations in DErPC (J. A. Killian et al., Biochemistry 1996, 35, 1037).