Cholesterol-Ceramide Interactions in Phospholipid and Sphingolipid Bilayers As Observed by Positron Annihilation Lifetime Spectroscopy and Molecular Dynamics Simulations.

Free volume voids in lipid bilayers can be measured by positron annihilation lifetime spectroscopy (PALS). This technique has been applied, together with differential scanning calorimetry and molecular dynamics (MD) simulations, to study the effects of cholesterol (Chol) and ceramide (Cer) on free volume voids in sphingomyelin (SM) or dipalmitoylphosphatidylcholine (DPPC) bilayers. Binary lipid samples with Chol were studied (DPPC:Chol 60:40, SM:Chol 60:40 mol ratio), and no phase transition was detected in the 20-60 °C range, in agreement with calorimetric data. Chol-driven liquid-ordered phase showed an intermediate free volume void size as compared to gel and fluid phases. For SM and SM:Cer (85:15 mol:mol) model membranes measured in the 20-60 °C range the gel-to-fluid phase transition could be observed with a related increase in free volume, which was more pronounced for the SM:Cer sample. MD simulations suggest a hitherto unsuspected lipid tilting in SM:Cer bilayers but not in pure SM. Ternary samples of DPPC:Cer:Chol (54:23:23) and SM:Cer:Chol (54:23:23) were measured, and a clear pattern of free volume increase was observed in the 20-60 °C because of the gel-to-fluid transition. Interestingly, MD simulations showed a tendency of Cer to change its distribution along the membrane to make room for Chol in ternary mixtures. The results suggest that the gel phase formed in these ternary mixtures is stabilized by Chol-Cer interactions.

Water-promoted hydrolysis of a highly twisted amide: rate acceleration caused by the twist of the amide bond.

The water-promoted hydrolysis of a highly twisted amide is studied using density functional theory in conjunction with a continuum dielectric method to introduce bulk solvent effects. The aim of these studies is to reveal how the twisting of the C-N bond affects the neutral hydrolysis of amides. To do so, both concerted and stepwise mechanisms are studied and the results compared to the ones from the hydrolysis of an undistorted amide used as reference. In addition, an extra explicit water molecule that assists in the required proton-transfer processes is taken into account. Our results predict important rate accelerations of the neutral hydrolysis of amides when the C-N bond is highly twisted, the corresponding barrier relaxation depending on the specific reaction pathway and transition state involved. Moreover, our calculations strongly suggest a change in reaction mechanism with degree of amide bond twist, and clearly point to a concerted mechanism at neutral pH for the hydrolysis of highly twisted amides. In addition, the twisting of the amide bond also provokes a higher dependence on an auxiliary water molecule for the concerted mechanism, due to the orthogonality of the lone pair of the nitrogen and the carbonyl pi orbital. There is a direct implication of these findings for biological catalytic mechanism of peptide cleavage reactions that undergoes ground-state destabilization of the peptide.