The composition and chemical structures of the membrane forming moleculesdetermine all essential properties of nature’s most important interface.Cellular membranes are subject to a hierarchical domain organization on the mesoscopiclength scale.^[1] This is triggered bypreferential interactions between the major constituents of the membrane, e.g.phospholipids, cholesterol (Chol), glycolipids, and membrane proteins.^[2] In particular, the attractiveinteractions between Chol and sphingolipids or saturated phospholipids and/or membraneproteins have been identified as the driving force for membrane domainformation.^[3] Key to the lipidphase separation phenomena is the preferential interaction of Chol with saturated lipidchains.^[4] Numerous examples ofthe specific interaction between Chol and saturated phospholipids or sphingomyelin havebeen reported.^[5] The interactionbetween Chol and saturated lipid chains is thought to originate by attractive van derWaals interactions between Chol’s planar and rigid ring system (particularly thesmooth α-face) and the saturated phospholipid chains.^[6] This leads to a stiffening of the lipid chains, asindicated by an order increase, which is referred to as area condensation.^[7] This increased packing densitydecreases membrane permeability.^[8]Surprisingly, the influence of the aliphatic Chol side chain on lipid condensation andmembrane domain formation is still not fully understood. Therefore, we havesystematically studied the influence of the methyl-branched(iso-series) side chain of Chol with 5 to 14 carbon atoms^[9] (Figure1A) on important membrane properties.

To evaluate the influence of the Chol side chain on the area condensation effect,²H NMR order parameters^[4] were measured for mixtures of either fully saturatedDPPC-d₆₂ or monounsaturatedPOPC-d₃₁ with 20 mol% of the respective sterolanalog (²H NMR spectra and order parameter plots are shown in supplementaryFigures S1 – S4).Pure lipid membranes are used as a reference featuring lowest chain order. SaturatedDPPC shows higher order parameters than the monounsaturated POPC. All sterols studiedinduce an increase in lipid chain order, i.e. condensation but the degree ofcondensation varies as a function of the length of theiso-branchedside chain. These differences are best displayed in the plot of the average orderparameters <S> of the phospholipid in the presence ofthe sterols with varying length of the side chain (Figure1B). There is a gradual increase in the POPC chain order fromandrost-5-en-3-β-ol (androstenol), which lacks a side chain, toi-C5-sterol, native Chol, andi-C10-sterol, whilethe longer chaini-C12- andi-C14-sterol analogs showdecreasing condensation again. Most interestingly is the fact that the sterol ringsystem of Chol is only responsible for ~40% of the lipid condensation asdemonstrated for androstenol, while thei-C8 side chain of Cholaccounts for ~60% of the condensation. Furthermore, it is remarkablethat native Chol induces a slightly lower lipid condensation thani-C10-sterol; for the latter, the lipid condensation is 6%higher.

For DPPC mixtures, highest chain order is observed in the presence of Chol, andthe attenuation of the ordering effect induced by the longer side chain sterol analogsis a bit attenuated. In contrast to the monounsaturated mixtures, thei-C8 side chain of Chol only accounts for ~40% of thecondensation in DPPC.

Changes in lipid chain order mostly affect the upper chain part, where anincrease in order between 40% (POPC) and 50% (DPPC) due to Chol isobserved. This can be evaluated from the difference order parameter plots shown inFigures S2 and S4. The increasein molecular order of the lower half of the chain much depends on the side chain lengthof the sterol analog and is optimal only for cholesterol or thei-C10sterol. Order increase leads to an increase in lipid chain length in the presence ofeach sterol analog, which is reported inTable S1.

Next, we investigated the influence of the sterol molecules on basic lipidmembrane properties. First, the main phase transition temperature of DPPC in thepresence of each sterol analog was investigated by differential scanning calorimetry(Figures S5 and S6). In thepresence of 20 mol% Chol, the phase transition temperature of DPPC increasesfrom 40.9°C to 41.5°C. In the presence of the sterol analogs, the phasetransition temperature decreased to well below 40°C for all sterols and forandrostenol it dropped to 30.5°CFigure S6).

Furthermore, the lateral diffusion of DPPC and the sterols was measured using¹H MAS PFG NMR (FigureS7).^[10] While lipiddiffusion rates can also be measured by fluorescence correlationspectroscopy,^[11] the NMRtechnique measures the diffusion rates of both phospholipid and the sterol in the samemixture without the need for fluorescence labels that may influence lipid diffusionrates. Diffusion coefficients determined for the DPPC and the sterols are shown inFigure 2. Within experimental error, DPPC and thevarious sterol molecules show approximately the same diffusion coefficients (filled barsrepresent DPPC, open bars the sterol). The diffusion coefficients ofi-C5-,i-C8-, andi-C10-sterols arerather similar and so are the diffusion coefficients for the DPPC in the presence ofthese sterols. However, for androstenol as well as fori-C12- andi-C14-sterols much larger diffusion coefficients are found, whichare even larger than for DPPC in the absence of Chol.

We also determined the diffusion coefficients of POPC in mixture with therespective sterol analog. Since POPC is not available in the chain perdeuterated form,signal superposition in the aliphatic region prevented the determination of thediffusion of the sterol and only POPC diffusion coefficients are reported inFigure S8. POPC showed thelargest diffusion coefficients. In the presence of androstenol,i-C5-,andi-C8-sterol POPC diffusion is slower with relatively similardiffusion coefficients, while the diffusion coefficients of POPC in the presence ofsterolsi-C10,i-C12, andi-C14decreased again significantly.

We also assessed the permeability of POPC and DPPC membranes in the presence ofthe respective sterols. A simple fluorescence assay was used that detects the permeationof dithionite ion across membranes by measuring the reduction of NBD-PE fluorescence inlipid vesicles.^[12] The reductionkinetics showed two components: a very fast decay due to the reduction of the NBD-PE inthe outer leaflet of the bilayer and a slower decay, which reflects the reduction of theNBD-PE in the inner leaflet upon dithionite ion permeation (Figure S9).Figure S10 shows the rateconstants for dithionite ion permeation across the POPC and DPPC bilayers, which areobtained by fitting the curves to a bi-exponential equation. DPPC shows highestpermeability for dithionite ion, which is relatively close to the phase transitiontemperature, where the permeability is generally high.^[13] Permeability is lower in the presence of all sterolanalogs; the lowest permeation rate is measured for Chol, indicating that its structureis optimal for decreasing the membrane permeability. In the presence of a sterol with alonger or shorteriso-branched side chain or with androstenol, as wellas in the absence of any sterol, these permeation rates are clearly higher.

Finally, we studied the potential of sterols with varying side chain lengths toinduce phase separation and the formation of liquid ordered(l_o) and liquid disordered(l_d) domains in mixtures of DOPC,N-stearoylsphingomyelin (SSM) and the respective sterol.Figure S11 shows confocalfluorescence images^[14] of giantunilamellar vesicles (GUVs) composed of DOPC, SSM, and the respective sterol molecule ata 1:1:1 molar ratio. As seen from this overview, all sterols induced the formation oflarge lipid domains irrespective of the length of the side chain, as reported by thefluorescence of N-Rh-DOPE representing a well-established marker for thel_d phase of GUVs made of lipid raft mixtures. Therefore,this phase appears in red in the images, while thel_o phase,which is depleted of N-Rh-DOPE, appears in dark colors (Figure S11).^[15]

In spite of the fact that all sterols induced phase separation, the partitioningof the fluorophore varies as a function of the sterol side chain length. To quantifythis distribution ratio, we calculated the intensity ratio of N-Rh-DOPE inl_o andl_d phases accordingto^[16] (Figure 3, filled bars). Almost exclusive partitioning of N-Rh-DOPEinto thel_d domain was found for raft mixtures composed ofnative Chol (0.2% in thel_o phase), whilepartitioning was less pronounced for thei-C5- andi-C12-sterols (2.5% and 2.1% in thel_o phase, respectively). Fori-C10-sterol, thel_o domain partitionincreases again to 4% and is highest in raft mixtures in the presence ofi-C14-sterol (8%) and androstenol (10.6%).Interestingly, the average domains covered approximately one-half of the line shown atthe particular cut made at the equator of the vesicle for all Chol analoges except forGUVs containing androstenol. Here thel_o domains is onlyabout 20% of the GUV. The only literature data available on sterols with longeror shorter side chain report thati-C4-sterol fails andi-C10-sterol promotes domain formation.^[17]

Finally, a rhodamine-labeled peptide (Rh-TMD), representing the transmembranedomain of Influenza A virus hemagglutinin, was incorporated at 1 mol% into theGUVs of the same mixtures.^[18] Thel_o/l_d distribution of thepeptide is shown inFigure 3 (open bars). In theternary mixtures composed of native Chol,i-C5-, ori-C10-sterol, Rh-TMD almost exclusively partitioned into thel_d domains (i.e., only 0.4%, 0.7%, and0.3% of the peptide were found in thel_o domain,respectively). In contrast, in mixtures containingi-C12- toi-C14-sterol, the amount of Rh-TMD found in thel_o domain increased to 2.6% and 3.7%,respectively. The partition equilibrium of the peptide was severely disturbed formixtures containing androstenol, where 10% of the peptide was found in thel_o domain, which is 25 times more than in thel_o domain of GUVs prepared with Chol.

Sterols are typical lipids of eukaryotic cells. While Chol represents the sterolin the plasma membranes of all animal cells, a number of different phytosterols arerepresentative for plants, while ergosterol represents the sterol in fungi and yeast.All these molecules feature a side chain at C-17 that consists of carbons 20 to 27 (seeFigure 1A). Phytosterols often have a methyl orethyl branch at C-24, and the number and position of double bonds in the side chainvaries. However, the overall length of theiso-branched side chainremains identical in the different sterols. In contrast to these differences in thesterols, the fatty acid composition of the cell membranes of plants, fungi, yeasts, andanimals is rather similar. Typically, the lipids in most of these membranes (mostlyphospho- and sphingolipids, but also glycolipids) contain~80–90% lipid chains of 16 or 18 carbons that are eithersaturated or contain one to three cis double bonds.^[19] Thus the majority of cellular membranes have a thickness thatmatches well with the molecular dimension of a sterol with aniso-branched side chain consisting of 8 carbons.

Several studies have demonstrated that even small changes in Chol’stetracyclic structure can have a profound impact on the lipid condensationeffect^[6b,20] and membrane domain formation.^[17,21] But contrary to the assumption that the interaction of Chol withmembrane phospholipids is based on interactions between the tetracyclic ring system ofthe former and the lipid chains of the latter molecules, we found that a major part ofthe condensation effect and the lateral organization of the lipids in raft mixtures areattributed to the Chol side chain. The side chain of Chol contributes~60% of the condensation in monounsaturated POPC membranes and~40% in saturated DPPC membranes. This suggests that the Chol side chainis crucial for especially the interaction with unsaturated phospholipids, whichrepresent the majority in biological membranes. Also, the lateral lipid diffusion andmembrane permeability appears to be optimized for Chol’si-C8side chain. Our biophysical data suggest that the Chol side chain has been fine tuned inevolution for an optimal interaction with phospholipids in lipid bilayers.

Evolutionary, sterols were introduced at a relatively late stage because of thelack of molecular oxygen for biosynthesis.^[8a] The evolutionary advantage of adding Chol to eukaryotic cellularmembranes was to modulate and refine membrane properties. In particular, the barrierfunction of the membrane is markedly improved.^[22] In that regard, it was not sufficient to develop a sterollacking side chain such as androstenol. As our study shows, only 40% of thetotal ordering of monounsaturated membranes (and concomitant denser packing) is achievedby the rigid tetracyclic carbon skeleton, and an important contribution comes from thealiphatic side chain. The additional van der Waals attraction between the Chol sidechain and the lower part of the lipid chains leads to an optimal increase in packingdensity of the lipids and thus the improved barrier function of the membrane (Figure S2). Membrane permeationis most restricted for native Chol, and even small alterations in the side chain lengthlead to a significantly impaired barrier function. Althoughi-C10-sterol induced a higher lipid condensation in POPC and anincreased lipid chain ordering particularly in the lower half of the chain (Figure S2), the membranepermeation for dithionite ion is much larger for the sterol with the longer sidechain.

The basis for the length of the Chol side chain is set in the biosynthesis ofthe molecule from dimethylallyl pyrophosphate and isopentenyl pyrophosphate in theisoprenoid metabolic pathway yielding squalene.^[23] This is the precursor for Chol and phytosterols and explainsthe general similarity between these molecules and in particular their aliphatic sidechains. From squalene, lanosterol evolved, which features a relatively rough surface dueto three additional methyl groups on the tetracyclic ring system(4-α-CH₃, 4-β-CH₃, and 14-CH₃), notpresent in Chol. This suggests that in the last steps of Chol evolution, the smoothnessof the α-face was optimized and the sterol-induced ordering of the lipid chainswas increased: in the presence of 20 mol% lanosterol, the average chain order ofPOPC membranes is 0.202, while in the presence of 20 mol% Chol it is0.206.^[24] This finalevolutionary steps resulted in a ~2% increase in lipid chain order.However, as our study on the length of the Chol side chain suggests, a very importantevolutionary step was the development of the general architecture of the sterolstructure featuring theiso-branched octyl side chain of themolecule.

In summary, our work emphasizes the crucial role of the Chol side chain for anumber of basic membrane properties as well as the lateral organization of the membranelipids and proteins. By a systematic analysis of sterols with varyingiso-branched aliphatic chains, we found that the side chain of Cholhas a profound impact on all of these properties. Typically, the characteristicproperties of Chol cannot be reproduced by molecules harboring a longer or shorter sidechain. This highlights the importance of the allegedly disordered Chol side chain forintermolecular interactions and membrane properties with significant consequences forthe cell biological role of the sterol.