Using ergosterol to mitigate the deleterious effects of ethanol on bilayer structure

Abstract

In wine fermentations, yeast is exposed to concentrated ethanol solutions. Ergosterol, a sterol that is found in lower eukaryotic membranes, helps preserve the structural integrity of yeast membranes in stressful environmental conditions. A premature arrest in ethanol production due to unknown metabolic changes in yeasts results in undesirably large concentrations of residual sugar and may be caused by the formation of an ethanol-induced interdigitated phase. We use atomistic molecular dynamics simulations to examine the induction of the interdigitated phase in model yeast membranes that contain either 0, 10, 20, 25 mol % ergosterol in ethanol concentrations of 0, 10, 15 vol %. The 25 mol % ergosterol system shows a similar level of interdigitation for the 0 and 10 vol % ethanol solutions, indicating that ergosterol molecules in this system are able to effectively counteract the disruptive behavior of ethanol molecules. However, at a 15 vol % ethanol solution, the amount of interdigitation triples and this ethanol concentration is similar to the concentrations found in stuck fermentations. The other three ergosterol concentrations studied (0, 10, 20 mol %) show larger quantities of interdigitation in the 10 vol % ethanol solution than the 0 vol % solution. Thus, the 25 mol % ergosterol bilayer, which is representative of the ergosterol concentrations seen in yeast membranes, is unique in the systems examined in its ability to delay the onset of ethanol-induced interdigitation. The concentration of ergosterol affects the permeability of a fluid-phase bilayer, where the 10 mol % ergosterol bilayer is more permeable to ethanol than either a bilayer containing no ergosterol molecules or a bilayer containing 20/25 mol % ergosterol. This lipid permeability appears to be correlated with the existence of a lipid region whose lipids neither have direct contact with ergosterol molecules nor exhibit bulk lipid/lipid interactions.

Figure 1

Attached to the DPPC phosphorus atom (P) is oxygen atom O10. Carbon atom CA is in the DPPC glycerol group, oxygen atom O16 is in the sn − 2 carbonyl group and oxygen atom O35 is in the sn − 1 carbonyl group. The sn − 1 carbonyl atoms are closer to the bilayer center than the sn − 2 carbonyl atoms. The oxygen atom in the ergosterol hydroxyl group is labeled O1 and the two double bonds and methyl groups that differentiate ergosterol from cholesterol are labeled 7, 22, and 24.

Figure 2

Density profiles for DPPC and ergosterol systems 25-0, 25-10, and 25-15 at T = 325 K.

Figure 3

The RDF between ergosterol atom O1 and DPPC atom O10 and O16 for system 10-0₃₂₅. The prominent RDF is for DPPC atom O10 and the fiure inset is the RDF for DPPC atom O16.

Figure 4

The RDF between the ethanol hydroxyl oxygen atom and DPPC atom O10 and O16 for system 10-0₃₂₅.

Figure 5

The DPPC sn − acyl chain order parameters for T = 298 K.

Figure 6

The relative increase in sn-2 S_CD resulting from inclusion of ergosterol at (a) 0 vol%, (b) 10 vol%, and (c) 15 vol% ethanol.

Figure 7

The relative decrease in sn-2 S_CD for 0, 10, and 15 vol% ethanol.

Figure 8

A picture of system 20-15 at T = 298 K. Interdigitation can be seen between the top leaflet lipids (white) and the bottom leaflet lipids (dark gray). Ergosterol molecules are light gray and ethanol molecules are black.

Figure 9

The density profies for 10-0₂₉₈ and 10–15₂₉₈. The squares and circles show interdigitated regions that exist in the 15 vol% system and not in the 0 vol% system. The black squares represent lipid density from the top leaflet (0–3 nm) found in the bottom leaflet and the gray circles represent lipid density from the bottom leaflet found in the top leaflet.

Figure 10

Interdigitated area (10⁻⁹ kg/m²) from the density profile curves at T = 298 K. The likelihood of finding the interdigitated phase in a system increases with area.

Figure 11

The DPPC sn − 2 acyl chain order parameters for T = 298 K and T = 325 K.

Figure 12

Ethanol molecules are assigned to Bins 1–5 based on the distance in xy between ethanol and ergosterol atoms. The black center represents an ergosterol molecule. Any partitioned ethanol molecule that has a distance <0.75 nm from an ergosterol atom is assigned to Bin 1. If the distance between the partitioned ethanol molecule and the nearest ergosterol atom is greater than 0.75 nm, it is assigned to Bin 2 – Bin 5. Bin distance specifications: Bin 1 (<0.75 nm) : Gray; Bin 2 (0.75≤1.0) : White; Bin 3 (1.0≤1.25) : Brick Patterned; Bin 4 (1.25≤1.5) : Dots; Bin 5 (1.5<) : Horizontal Lines. Note that since the lipid area is dependent on both temperature and ethanol/ergosterol concentration, the number of bins that exist vary by system.

Figure 13

The fraction of the total area occupied by each bin at T = 298 K for systems 10-0, 20-0, and 25-0. Bin specifications and corresponding colors are described in Figure 12.

Figure 14

The fraction of the total area occupied by each bin at T = 325 K.

Figure 15

The fraction of partitioned ethanol molecules in each bin/bin area (T = 298 K).

Figure 16

The fraction of partitioned ethanol molecules found in each bin/bin area (T = 325 K).