ESR STUDIES ED SPIN-LABELED MEMBRANES ALIGNED BY ISOPOTENTIAL SPIN-DRY ULTRACENTRIFUGATION - LIPID-PROTEIN INTERACTIONS

Electron spin resonance (ESR) studies have been performed on spin-labe led model membranes aligned using the isopotential spin-dry ultracentr ifugation (ISDU) method of Clark and Rothschild. This method relies on sedimentation of the membrane fragments onto a gravitational isopoten tial surface with simultaneous evaporation of the solvent in a vacuum ultracentrifuge to promote alignment. The degree of alignment obtainab le using ISDU, as monitored by ESR measurements of molecular ordering for both lipid (16-PC) and cholestane spin labels (CSL), in dipalmitoy lphosphatidylcholine (DPPC) model membranes compares favorably with th at obtainable by pressure-annealing. The much gentler conditions under which membranes may be aligned by ISDU greatly extends the range of m acroscopically aligned membrane samples that may be investigated by ES R. We report the first ESR study of an integral membrane protein, bact eriorhodopsin (BR) in well-aligned multilayers. We have also examined ISDU-aligned DPPC multilayers incorporating a short peptide gramicidin A' (GA), with higher water content than previously studied. 0.24 mol % BR/DPPC membranes with CSL probe show two distinct components, prima rily in the gel phase, which can be attributed to bulk and boundary re gions of the bilayer. The boundary regions show sharply decreased mole cular ordering and spectral effects comparable to those observed from 2 mol % GA/DPPC membranes. The boundary regions for both BR and GA als o exhibit increased fluidity as monitored by the rotational diffusion rates. The high water content of the GA/DPPC membranes reduces the dis ordering effect as evidenced by the reduced populations of the disorde red components. The ESR spectra obtained slightly below the main phase transition of DPPC from both the peptide- and protein-containing memb ranes reveals a new component with increased ordering of the lipids as sociated with the peptide or protein. This increase coincides with a b road endothermic peak in the DSC, suggesting a disaggregation of both the peptide and the protein before the main phase transition of the li pid. Detailed simulations of the multicomponent ESR spectra have been performed by the latest nonlinear least-squares methods, which have he lped to clarify the spectral interpretations. It is found that the sim ulations of ESR spectra from CSL in the gel phase for all the lipid me mbranes studied could be significantly improved by utilizing a model w ith CSL molecules existing as both hydrogen-bonded to the bilayer inte rface and non-hydrogen-bonded within the bilayer.