Mt. Ge et al., ESR STUDIES ED SPIN-LABELED MEMBRANES ALIGNED BY ISOPOTENTIAL SPIN-DRY ULTRACENTRIFUGATION - LIPID-PROTEIN INTERACTIONS, Biophysical journal, 67(6), 1994, pp. 2326-2344
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.