3 research outputs found
Atomic Force Microscopy Characterization of Palmitoylceramide and Cholesterol Effects on Phospholipid Bilayers: A Topographic and Nanomechanical Study
Supported
planar bilayers (SPBs) on mica substrates have been studied
at 23 °C under atomic force microscopy (AFM)-based surface topography
and force spectroscopy with two main objectives: (i) to characterize
palmitoylceramide (pCer)-induced gel (L<sub>β</sub>) domains
in binary mixtures with either its sphingolipid relative palmitoylsphingomyelin
(pSM) or the glycerophospholipid dipalmitoylphosphorylcholine (DPPC)
and (ii) to evaluate effects of incorporating cholesterol (Chol) into
the previous mixtures in terms of Cer and Chol cooperation for the
generation of lamellar gel (L<sub>β</sub>) phases of ternary
composition. Binary phospholipid/pCer mixtures at <i>X</i><sub>pCer</sub> < 0.33 promote the generation of laterally segregated
micron-sized pCer-rich domains. Their analysis at different phospholipid/pCer
ratios, by means of domain thickness, roughness, and mechanical resistance
to tip piercing, reveals unvarying AFM-derived features over increasing
pCer concentrations. These results suggest that the domains grow in
size with increasing pCer concentrations while keeping a constant
phospholipid/pCer stoichiometry. Moreover, the data show important
differences between pCer interactions with pSM or DPPC. Gel domains
generated in pSM/pCer bilayers are thinner than the pSM-rich surrounding
phase, while the opposite is observed in DPPC/pCer mixtures. Furthermore,
a higher breakthrough force is observed for pSM/pCer as compared to
DPPC/pCer domains, which can be associated with the preferential pCer
interaction with its sphingolipid relative pSM. Cholesterol incorporation
into both binary mixtures at a high Chol and pCer ratio abolishes
any phospholipid/pCer binary domains. Bilayers with properties different
from any of the pure or binary samples are observed instead. The data
support no displacement of Chol by pCer or vice versa under these
conditions, but rather a preferential interaction between the two
hydrophobic lipids
Solvation and Hydration of the Ceramide Headgroup in a Non-Polar Solution
The microscopic hydration of the
ceramide headgroup has been determined
using a combination of experimentalboth NMR and neutron diffraction
techniques and computational techniquesempirical potential
structure refinement (EPSR) and molecular dynamics (MD). The addition
of water to ceramide in chloroform solutions disrupts the chloroform
solvation of the ceramide headgroup, and the water forms distinct
pockets of density. Specifically, water is observed to preferentially
hydrate the two hydroxyl groups and the carbonyl oxygen over the amide
NH motif. Further assessment of the location and orientation of the
water molecules bound to the ceramide headgroup makes it clear that
the strongly solvated carbonyl moiety of the amide bond creates an
anchor from which water molecules can bridge via hydrogen bonding
interactions to the hydroxyl groups. Moreover, a significant difference
in the hydration of the two hydroxyl groups indicates that water molecules
are associated with the headgroup in such a way that they bridge between
the carbonyl motif and the nearest neighbor hydroxyl group
Fluorescent Polyene Ceramide Analogues as Membrane Probes
Three
ceramide analogues have been synthesized, with sphingosine-like
chains containing five conjugated double bonds. Pentaene I has an <i>N-</i>palmitoyl acyl chain, while the other two pentaenes contain
also a doxyl radical, respectively, at C5 (Penta5dox) and at C16 (Penta16dox)
positions of the <i>N</i>-acyl chain. Pentaene I maximum
excitation and emission wavelengths in a phospholipid bilayer are
353 and 478 nm, respectively. Pentaene I does not segregate from the
other lipids in the way natural ceramide does, but rather mixes with
them in a selective way according to the lipid phases involved. Fluorescence
confocal microscopy studies show that when lipid domains in different
physical states coexist, Pentaene I emission is higher in gel than
in fluid domains, and in liquid-ordered than in liquid-disordered
areas. Electron paramagnetic resonance of the pentaene doxyl probes
confirms that these molecules are sensitive to the physical state
of the bilayer. Calorimetric and fluorescence quenching experiments
suggest that the lipids under study orient themselves in lipid bilayers
with their polar moieties located at the lipid–water interface.
The doxyl radical in the <i>N</i>-acyl chain quenches the
fluorescence of the pentaene group when in close proximity. Because
of this property, Penta16dox can detect gel–fluid transitions
in phospholipids. The availability of probes for lipids in the gel
phase is important in view of novel evidence for the existence of
gel microdomains in cell membranes