Table 3. RP-1 interaction with lipid systems by quantitative FTIR spectroscopy.
Lipid System | pH | % Conformation | Tilt Angle Θ | KA (M−1) | KD (M) | |||
α-helix | loop-turn | β-sheet | disordered | |||||
POPE∶POPG | 7.5 | 51.5 | 31.6 | 6.5 | 10.4 | 51° | 5.3×104 | 1.2×10−7 |
POPE∶POPG | 5.5 | 50.2 | 32.9 | 8.7 | 8.2 | 46° | 1.3×104 | 9.2×10−8 |
POPC∶CHO | 7.5 | 42.9 | 33.8 | 13.2 | 10.1 | 34° | 0.3×104 | 5.3×10−7 |
POPC∶CHO | 5.5 | 40.2 | 34.2 | 14.9 | 10.7 | 31° | 0.1×104 | ND |
The secondary structure and orientation of RP-1 was determined using representative bacterial (POPE/POPG; mole ratio 3∶1) and eukaryotic (POPC/cholesterol; mole ratio 1.2∶1) mimetic liposomal systems. For conformation assessment lipid-peptide films were dispersed in deuterated buffer (10 mM PIPES pH 5.5 or 10 mM HEPES pH 7.5) and FTIR spectra of the samples were averaged for 256 scans at a gain of 4 and a resolution of 2 cm−1. The relative amounts of α-helix, β-turn, β-sheet, and random (disordered) structures were estimated by Fourier self-deconvolution and the tabulated results represent means from four independent and highly reproducible determinations for each environment SE 5%, or better. Kassoc was measured by introducing RP-1 to solutions containing large unilamellar vesicles (10 mM PIPES pH 5.5 or 10 mM HEPES pH 7.5). The orientation of the RP-1 peptide in the lipid bilayer of eukaryotic and bacterial membrane mimetic systems was determined using polarization (0° to 90°) to determine the insertion, or tilt angle of the peptide helical axis in the lipid multilayers. The binding of the peptide to lipid was expressed as an association constant Kassoc [1], where [P] is the molar concentration of RP-1 peptide in solution, [L] is the molar concentration of lipid and [PL] is the molar concentration of peptide bound to lipid [39].