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1 | SCIENCE ADVANCES RESEARCH ARTICLE BIOPHYSICS Copyright © 2019 The Authors, some rights reserved; Entropic effects enable life at extreme temperatures exclusive licensee American Association 3 1 1 2 1 *, Karthik Diraviyam , , Takaoki Koyanagi Young Hun Kim , Kaifu Gao *, Geoffray Leriche for the Advancement 4 4 1 5 6 David Onofrei , Gregory P. Holland , Anirvan Guha , , Nathan Gianneschi , Joseph Patterson of Science. No claim to † 3 5 2 1 original U.S. Government , Michael Mayer , David Sept , Jerry Yang Michael K. Gilson Works. Distributed under a Creative Maintaining membrane integrity is a challenge at extreme temperatures. Biochemical synthesis of membrane- Commons Attribution spanning lipids is one adaptation that organisms such as thermophilic archaea have evolved to meet this challenge NonCommercial and preserve vital cellular function at high temperatures. The molecular-leve l details of how these tethered lipids License 4.0 (CC BY-NC). affect membrane dynamics and function, however, remain unclear. Using synthetic monol ayer-forming lipids with ng makes membrane permeation an entropically controlled transmembrane tethers, here, we reveal that lipid tetheri process that helps to limit membrane leakage at elevated te r-forming lipid membranes. mperatures relative to bilaye ew that permeation through membranes made of tethered All-atom molecular dynamics simulations support a vi s to tighter lipid packing, providing a molecular interpre- lipids reduces the torsional entropy of the lipids and lead tation for the increased transition-state entropy of leakage. Downloaded from INTRODUCTION extracts ( 3 ) and the lack of available synthetic bipolar tethered lipids, ). 1 Thermophilic archaea can live at temperatures exceeding 90°C ( there are few reported studies that examine the effect of lipid tethering lipid membranes Among other adaptations, these organisms generate on membrane function at elevated temperatures. For instance, previous with unique structural features that help retain their barrier function in reports show that tethering of synthetic lipids decreases membrane – 5 ). Specifically, archaeal lipids ( these extreme environments ( 4 )have 2 leakage of small molecules and ions at high temperatures compared http://advances.sciencemag.org/ ether linkages between their polar head groups and lipid tails, rather 16 , 15 , 3 to untethered lipids ( ). While these reports provided important than the more labile ester groups found in eukaryotic and prokaryotic first steps toward examining the pro perties of membranes containing ). In addition, archaea lipids typically contain highly 6 lipid membranes ( xtures or lack of control of relevant tethered lipids, their use of lipid mi branched lipid tails, unlike non-archaeal lipids; these phytanyl chains nsition temperatures, distribution lipid characteristics such as phase tra 7 ) while simultaneously maintaining improve the packing of the lipids ( of sizes of liposomes, or lamellarity makes it difficult to conclude any the lipids in a liquid phase (as opposed to a gel phase) over a wide range mechanistic relationship between the structure of bipolar tethered lipids “ ” archaeal lipids can be of temperatures ( 6 ). Therefore, heat-tolerant and membrane leakage profiles at different temperatures. Here, we report used at both high and low temperatures because of their liquid crystal- a controlled permeation analysis of temperature-dependent liposome line phase and low permeability at a wide range of temperatures. Addi- leakage for three synthetic bipolar tethered or monopolar untethered , 9 tional structural elements ( ) commonly found in archaeal lipids, in 8 lipids that, in combination with molecular dynamics (MD) simulations, ), may also impart important functional 10 particular lipid tethering ( provides a molecular description for the effect of lipid tethering on on May 5, 2019 characteristics in archaeal membranes; however, the relationship be- membrane leakage at temperatures ranging from 22° to 70°C. tween these structural features and temperature-dependent membrane integrity remains poorly understood. RESULTS One of the most striking features of membranes from thermophilic Lipid tethering reduces temperature-dependent archaea is that, unlike eukaryotic cells, they contain bipolar tethered membrane leakage that is, fully membrane-spanning lipids containing two polar lipids — To examine the effect of lipid tethering on membrane leakage as a head groups and covalently linked lipid tails that form monolayer ulated carboxyfluorescein (CF) at function of temperature, we encaps membranes — as opposed to the monopolar phospholipid bilayers self-quenching concentration (100 mM) in three different thermostable of eukaryotic cells. It has been shown that the fraction of bipolar (fig. S1G) liposomal formulations, composed of pure synthetic lipids tethered lipids in archaeal membranes increases with higher tem- with ether linkages (T32, T36, and U16; Fig. 2A and fig. S1A), and used ), leading to speculation that molecular 14 11 – peratures (Fig. 1) ( fluorescence intensity measurements to monitor the efflux of CF at tem- tethering of lipids plays a role in regulating flexibility and fluidity of peratures of 22°, 37°, 50°, 60°, and 70°C over the course of 8 hours. Here, archaeal membranes at elevated temperatures ( 13 ). However, due to T32 is a hemicyclic bipolar lipid with a tethered chain length of 32 meth- the complexity of mixtures of natural lipids present in membrane ylene units; monopolar U16 lipid is effectively T32 cut in half at the C16/C17 bond of the tethered lipid chain, and T36 is the same as 1 Department of Chemistry and Biochemistry, University of California, San Diego, 2 T32 except that the tethered chain is four methylene groups longer Department of Biomedical Engineering, Center for Com- La Jolla, CA 92093, USA. putational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI and, thus, makes it possible to probe the effect of a longer tether on 3 Skaggs School of Pharmacy and Pharmaceutical Sciences, University 48109, USA. membrane permeation. We used CF in these elevated temperature 4 of California, San Diego, La Jolla, CA 92093, USA. Department of Chemistry and 5 studies because we found that the rate of CF leakage could be readily Biochemistry, San Diego State University, San Diego, CA 92182, USA. Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, Fribourg, Switzerland. opically across all temperatures. and reproducibly monitored spectrosc 6 Departments of Chemistry, Materials Science and Engineering, and Biomedical Figure 2 (B to D) plots the time-dependent efflux of CF from unilamellar Engineering, Northwestern University, Evanston, IL 60208, USA. liposomeswithadiameterof~80nm(figs.S1,BtoD,andS2A),com- *These authors contributed equally to this work. posed of each of the three pure lipids, as a function of temperature. † Corresponding author. Email: [email protected] et al Sci. Adv. 2019; 5 :eaaw4783 1 May 2019 1of8 Kim .,

2 | SCIENCE ADVANCES RESEARCH ARTICLE tether by four methylene groups fr omT32toT36doesnotaffectthe temperature-dependent kinetics of CF leakage, suggesting that the effects on membrane structure as a result of extending the length of the lipid tether do not play a dominant role in temperature-dependent leakage. MD simulations reveal that reduced local diffusion of tethered lipids correlates with reduced permeation To gain insight at the microscopic level into the effects of tethering on permeation, we performed MD simulations of membranes constructed from each of the three lipids (U16, T32, and T36). In these simulations, all three lipids formed stable memb ranes at 300, 315, and 330 K (fig. S5), and the results provided estimates for parameters such as membrane nd self-diffusion constants of lipids, thickness, bending stiffness, lateral a average area per lipid, and variance in area per lipid at these different temperatures. To test the validity of the simulations, we compared the experimental and calculated values for the thickness of the membranes Fig. 1. Correlation between the abundance of tethered lipids and growth tem- 10 )be- (Fig. 3A and fig. S6) and the rank o rder of the lateral diffusion ( perature in archaea. The percentage of tethered lipids in the membranes of three tween the lipids (Fig. 3B and figs. S1E and S7) at 300 K. These values different archaea organisms was found to increase with increasing external Downloaded from were consistent between calculations and experimental measurements, growth temperature ( 14 – 12 ). ns represent the lipids and mem- which suggest that the MD simulatio branes under investigation well. While the rate constants (and permeability coefficients) for CF leak- We conducted simulations at different temperatures to provide ageoutofliposomesfromallthreelipidsincreasewithincreasingtem- insight into the effects of temperatu re on lipid dynamics. Previous stu- perature (Fig. 2A and fig. S3), liposomes composed of T32 and T36 et al dies carried out by Matsuno ) with extracted archaeal lipids 13 .( http://advances.sciencemag.org/ showed a reduced dependence on temperature compared to U16. suggest that lipid tethering influences membrane flexibility, while pre- Theonlydifferencebetweentheseleakageexperimentswasthepresence vious simulations show no correlation between bending stiffness and of the tether in the lipid, which has a substantial influence on the tem- 10 ). Nagle and colleagues also previously re- lipid tethering at 300 K ( perature dependence of CF leakage. A similar trend in CF leakage rates g that permeation correlates with ported computational results showin was observed when using ~200-nm-d iameter liposomes (figs. S2B and 18 ) or area per lipid with bilayer-forming lipids variance in lipid area ( S4), demonstrating that the observed effect of tethering persists at re- 19 ( ). The present MD simulations of U16, T32, and T36 show that both duced membrane curvature. the variance in lipid area and area per lipid increase with increasing tem- hat these two calculated parameters perature, as expected (fig. S8), and t Entropic effects from lipid tethering reduces permeation at are highly correlated with each other (Pearson correlation = 0.94, r < P 0.001; fig. S9) across all three lipids at all temperatures. While the MD elevated temperatures on May 5, 2019 To gain mechanistic insight into the temperature dependence of leak- simulations support the idea that T32 and T36 exhibit a smaller vari- age, we performed a Eyring-Polanyi ki netic analysis of the leakage data ance in lipid area at all temperatures compared to U16 (which suggests ( ). This analysis reveals the contri butions of the enthalpy and entropy 17 that lipid tethering leads to tighter lipid packing), we did not find a of activation for CF leakage from liposomes. A key finding is that the strong correlation between the experimental leakage rates of CF obtained r CF leakage from the bipolar teth- activation enthalpy and entropy fo at different temperatures and the calculated variance in area per lipid ered T32 and T36 lipids are significantly different ( < 0.0001) from P = 0.07; Fig. 3C) or area per lipid (Pearson correlation = 0.63, (Pearson r P those of the monopolar untethered U16 lipid (Fig. 2E). Specifically, r P correlation = 0.65, = 0.06; fig. S9) at 22°, 37°, and 60°C. tethering reduces (makes less positive) the enthalpy of activation To examine whether any other calculated parameters of the lipids ‡ 1 − (Fig. 2E), which on its ) of CF leakage by 3.6 to 4.7 kcal mol H D ( could be predictive of leakage properties for this set of lipids, we own would lead to increased leakage. However, tethering also decreases examined the correlation between observed leakage rates at different ‡ )ofCFleakageby S (makes more negative) the entropy of activation ( D correla- temperatures and the lateral (lo ng-range) diffusion (Pearson r − − 1 1 K , and this drop in the activation entropy leads to 12 to 16 cal mol r = 0.002; Fig. 3D) and self (local) diffusion (Pearson P tion = 0.87, cor- greater resistance of the tethered lipids to leakage at high temperature. < 0.0001; Fig. 3E) for all three synthetic lipids at three P relation = 0.98, Thus, the larger activation entropy of leakage through membranes of different temperatures (300, 315, an d 330 K). Here, the Pearson corre- the tethered lipids leads to an increasing Gibbs free energy of activation lation coefficients ( r ) reveal that the lipid self-diffusion was the better ‡ ) of CF leakage as temperature increases, rendering liposomes ( G D parameter for predicting permeability in this set of lipids. composed of bipolar tethered lipids more resistant to changes in leakage rates at temperatures in the range of 22° to 70°C compared to mono- CF in the membrane introduces a stronger reduction in polar untethered lipids (Fig. 2F). torsional entropy in tethered over untethered lipids Comparison of CF leakage from liposomes with diameters of ~80 or The experimental leakage studies show that tethering increases the en- ~200 nm revealed that the liposome size, and hence membrane curva- tropic penalty for passage of CF through the membrane and thus re- ‡ ‡ for CF leakage (Fig. 2E), and S D H ture, influences the magnitude of D duces permeation at elevated temperature. We speculated that the ‡ ‡ entropy differences between tethered and untethered lipids might re- H DD but the differences in thermodynamic values ( and S DD )between tethered and untethered lipids are essentially independent of liposome flect, at least in part, differences in how the presence of CF into the diameter (Fig. 2, G and H). Addition ally, increasing the length of the membrane influences the chain entropy of the lipids. We used MD ., Sci. Adv. 5 :eaaw4783 1 May 2019 2of8 et al Kim 2019;

3 | RESEARCH ARTICLE SCIENCE ADVANCES Downloaded from http://advances.sciencemag.org/ on May 5, 2019 ( A ) Structures of monolayer-forming tethered lipids T 32 and T36 and bilayer-for ming untethered lipid Fig. 2. Kinetic analysis of leakage of encapsulated CF from liposomes. to es are global fits ) Temperature-dependent CF release profile from liposomes prepared from T32 (B), T36 (C), and U16 (D). Data points show replicate results; solid curv D B U16. ( ‡ ‡ D H E to rate theory across all of the data in each graph simultaneously (see Materials and Methods). ( ) Calculated enthalpy ( D S ) and entropy ( ) of activation for encapsulated Kinetic analysis “ CFleakage fromliposomes using Eyring equation (see the section). Thermodynamic parameters were calculated using replicates from two separate liposome ” ) Effect of temperature on Gibbs free energy of activation for CF leakage through 80-nm liposomes. Gibbs free F preparations with different diameters (~80 and ~200 nm). ( ‡ ‡ ‡ ‡ D energy was calculated at different temperature (22°, 37°, 50°, 60°, and 70°C) using the Gibbs fundamental equation ( G T H D D S = ). ( G )Changeinenthalpy( DD H − ) for T32 ‡ ‡ between the two tethered lipids and between thedata from obtained for U16. Student ’ s t test revealed no significant differencein D H H D (green) and T36 (blue) compared to ‡ ‡ ) Change in entropy ( DD S H different liposome diameters (and, hence, membrane curvature). ( D S ) for T32 (green) and T36 (blue) compared to obtained for U16. simulations to test this idea, examining changes in the first-order ap- red lines). However, there is a noticeable drop in entropy for the bipolar 1 proximation of the chain entropy (S )( 20 ), when CF is situated partway tethered lipids (Fig. 4, B and C, solid versus dashed green lines), with the through one leaflet of a membrane consisting of bipolar tethered lipid largest reduction in torsional entropy occurring at carbon atoms near T32 or of monopolar untethered lipid U16, as depicted in a sample the center of the membrane. These effects are larger for the methylene snapshot from one simulation in Fig. 4A. chains than for the phytanyl chains, w hich are not tethered in either T32 The baseline torsional entropy i n the absence of CF (Fig. 4, B and C, or U16 (Fig. 4D). The entropies provided are per lipid, averaged over all dashedlines)issubstantiallylowerforthebipolartetheredlipids(Fig.4, lipids in the simulation (64 for the te thered and 128 for the untethered), B and C, green) than for the monopolar untethered lipids (Fig. 4, B and so the net effect across all lipids in the membrane is substantial. C, red), as may be expected due to the conformational constraint im- Although the CF is held in the top half of the membrane for these posed by tethering. The difference is greater for the tethered chain simulations (Fig. 4A), the changes in entropy are similar between the (Fig. 4B) than for the phytanyl chain (Fig. 4C). When CF is inserted top and bottom membrane leaflets, as evident from the symmetry of into the membrane, there is minimal change in the torsional entropy the perturbations around the centr al carbon torsion (torsion on carbon of the monopolar untethered lipids (Fig. 4, B and C, solid versus dashed ” top “ ” bottom “ and 13 in Fig. 4, B and C) and from the similarity of the ., Sci. Adv. 2019; 5 :eaaw4783 1 May 2019 3of8 et al Kim

4 | RESEARCH ARTICLE SCIENCE ADVANCES brane thickness measured experimentally by atomic force microscopy ) Correlation between mem A ( Fig. 3. Computational modelingof bilayerandmonolayermembranes. ) Correlation between variance in area per lipid determined C ) Lateral diffusion coefficient of lipids in the membrane determined by MD simulation. ( B (AFM) and MD simulations. ( = 0.07). The dashed lines represent the 95% ge rates obtained at different temperatures (Pearson by MD at different temperatures and experimental CF leaka P correlation = 0.63, r confidence band of the best-fit line. ( ) Correlation between lipid lateral diffusion determined by MD and e xperimental CF leakage rates at d ifferent temperatures (Pearson r D correlation = 0.87, P = 0.002). The dashed lines represent the 95% confidence band of the best-fit line. ( E ) Correlation between lipid self-diffusion determined by MD and ex- Downloaded from P correlation = 0.98, r perimental CF leakage rates at different temperatures (Pearson < 0.0001). The dashed lines represent the 95% confidence band of the best-fit line. http://advances.sciencemag.org/ on May 5, 2019 )usingGAFF( ( ) parameters, with TIP3P 36 ) and Lipid14 ( 35 Fig. 4. First-order entropy of lipid torsions. 34 ) Entropy analysis based on MD simulations with AMBER 14 ( D to A water, of membranes with and without CF restrained d in the T32 monolayer at a target depth in one leaflet in the presence of 150 mM KCl. (A) Simulation snapshot of CF restraine membrane (100 ns, 300 K, 64 lipids). (B and C) First-order entropy of lipid torsions as a function of torsion position, where torsion 1 is at the top of the membrane and torsion 26 is at the bottom (A). Results are shown here for the unbranched methylene chain (B) and phytanyl chain (C) of each lipid. Red, untethered (U16) lipids; green , tethered (T32) lipids; solid lines, rst-order entropy reported for each t 64instancesofthetorsioninthe CF present in the membrane; dashed lines, CF absent from the membrane. The fi orsion is a per lipid average over all 64 lipid molecules of the tethered membrane simulations or the 128 lipid molecules of the untet hered membrane simulations. (D) Effect of the presence of CF in the membrane on the − 1 1 − first-order torsional entropy (in cal mol K )perlipidofthesimulatedT32andU16lipidmembranes,partitionedbylipidchainandbilayerleaflet(topversusbottom). computed changes in entropy provid ed in Fig. 4D. Thus, in the tethered reduces membrane permeability at elevated temperatures relative f of the membrane propagate to case, CF ’ s interactions with the top hal to untethered lipids. Further analyses by MD simulations offer a the lower half, amplifying the entropic penalty for permeation, while in possible explanation for this observa tion, namely, that the bipolar teth- the untethered case there is no noticeable effect in either leaflet. ropy than do monopolar untethered ered lipids lose more torsional ent lipids when CF enters the membrane. Evidently, the addition of CF sy- nergizes with the baseline conformat ional restrictions imposed by the DISCUSSION tethers, leading to increased ordering and lower entropy. The experi- We demonstrate that lipid tethering significantly increases the en- mental observation of a reduced enthalpic barrier may be more difficult tropic barrier for membrane permeation of CF and, accordingly, to understand, but it is interesting to speculate about a possible analogy ., Sci. Adv. 2019; 5 :eaaw4783 1 May 2019 4of8 et al Kim

5 | RESEARCH ARTICLE SCIENCE ADVANCES with hydrophobic solvation. When a small hydrophobic molecule is chemical (i.e., hydrolytic) stability of lipids ( 6 ). We incorporated an un- s at its surface respond by forming placed in water, the water molecule posed to a saturated hydrocarbon tethered phytanyl acyl chain (as op cage-like, hydrogen-bonded structures characterized by low entropy ll lipids to reduce the probability chain without methyl groups) into a and low enthalpy. Placing CF into the membrane may similarly cause 6 of a phase transition in the temperature range of interest ( )andto the tethered lipids to adopt well-packed structures with favorable lipid- 29 avoid phase transition – induced leakage ( 27 – ). This design made it lipid interactions (low enthalpy) an d increased ordering (low entropy). possible to generate lipids T32, T36, and U16 in ~10 synthetic steps, Atthesametime,itshouldbenotedthattherearemanyotherentropic ). as previously described ( 10 and enthalpic differences between tethered and untethered lipids that could contribute to the overall effects observed experimentally. These Liposome preparation ifferences in translational and ori- contributions could arise, e.g., from d We prepared a liposome solution (10 mg/ml) by first dissolving 5 mg of ferences in orientational entropy of entational entropy of the lipids, dif lipid of interest into a 5-ml round bottom flask in a dichloromethane/ the CF molecule in the membrane, and differences in the correlations MeOH (7:3) solution. A thin lipid film was achieved by evaporating the among the various degrees of freedom. solvent using a rotary evaporato r (Buchi RE-111) and then by drying Simulations showed no significant correlation between tethering further with a high-vacuum pump ( Welch 1402) for 4 hours. The thin and membrane flexibility (est 10 imated by bending stiffness) ( ), as had lipid film was then hydrated at 10 mg/ml in either 10 mM Hepes buffer ). However, in accord wi th results from flu- 13 been proposed by others ( fered saline (PBS) (1×, pH 7.4), (150 mM KCl, pH 7.0), phosphate-buf orescence recovery after photobleaching experiments (fig. S1E), the or a solution of PBS containing 100 mM CF by vortexing the solution present MD simulations support a significant effect of lipid tethering for 30 s, followed by sonication in a water bath sonicator (Branson Downloaded from on the viscosity of the membrane, indicated by the diffusion coefficients 2510) for 30 min. After sonication, the lipid mixture underwent five of the lipids. Given that membrane viscosity is thought to be an impor- − freeze-thaw cycles that consisted of 2 min at 78°C, followed by 2 min ),andthatwefindthatentropy 21 tant determinant of permeability ( at 50°C. The liposomal suspension was then successively extruded plays an important role in the permeability of bipolar tethered versus 00- and 200-nm polycarbonate mem- (Avanti Mini Extruder) through 4 monopolar untethered lipid membrane s, it is of interest that the diffu- branes (51 times for each membran e) to generate ~200-nm-diameter sion constant of molecules in a liquid is expected to correlate with the liposomes. Smaller liposomes (~80 nm diameter) were produced using http://advances.sciencemag.org/ partial molar entropy ( 22 – 24 ). Intuitively, more freedom of motion additional extrusion with 100- and 50-nm polycarbonate membranes correspondstobothhigherentropyandhighermobility.Similarcon- (51 times for each membrane). Liposome radii are shown in fig. S2. siderations may apply in the context of a membrane. For CF-encapsulated liposomes, free CF was removed by gel filtra- In conclusion, this study reveals a fundamental principle for main- tion through a Sephadex G-100 column eluted with PBS (1×, pH 7.4), taining membrane integrity at elevat ed temperatures. Namely, to render and the lipid solution was then stored at 4°C in a Protein LoBind vated temperatures, it is helpful for membranes resistant to leakage at ele Eppendorf tube. the free energy barrier of molecular leakage to include a large entropy of activation. Lipid tethering is one molecular strategy toward achieving – Cryo electron microscopy imaging of liposomes this goal. l of liposomes in PBS (2 mg/ml, ~80 nm diameter by We applied 5 m DLS) to a holey grid, which had been previously treated by glow on May 5, 2019 discharged in an oxygen plasma chamber. The cryo – electron micros- MATERIALS AND METHODS copy (EM) sample was vitrified using liquid ethane as the cryogen. The General information frozen sample was transferred into a precooled cryo-transfer holder to Dynamic light scattering (DLS) measurements were performed on a maintain a low temperature. The image was acquired on a FEI Tecnai Wyatt DynaPro NanoStar (Wyatt Technology, Santa Barbara, CA) G2 Sphera microscope operated at 200 keV using a Gatan UltraScan instrument using a disposable cuvette (Eppendorf UVette, 220 to 1000 UHS 4 MP charge-coupled device camera. All three lipids 1600 nm), and data were processed using Wyatt DYNAMICS V7 soft- ly unilamellar liposomes by (U16, T32, and T36) can form most ware. Each analysis involved an average of 10 measurements. The data lipidfilmhydrationandextrusion through polycarbonate membranes were exported for final plotting using GraphPad Prism 5 (GraphPad (fig. S1, B to D). Software Inc., La Jolla, CA). Absorbance measurements were taken on a PerkinElmer EnSpire Differential scanning calorimetry multimode plate reader. Untreated Co rning 96-well half area black flat Suspensions of liposomes were prepared by sonication of lipid films for bottom polystyrene microp lates were used (ref. 3694). 30 min in ultrapure water. All liposome samples contained a final con- centration of ~5% lipid by weight. Differential scanning calorimetry (DSC) n duplicate using a Thermal Anal- experiments were performed i Lipid design and synthesis ysis Q2000 DSC. Each experiment in volved a 1°C/min ramp from 0° to As opposed to natural archaeal tethered lipids, which typically contain 80°C under dry helium at 50 ml/min. TA Universal Analysis was used 5 two transmembrane tethers to form a C-40 macrocyclic lipid ( ), we for these samples. DSC experiments revealed that all to extract the T synthesized and studied bipolar hemi-macrocyclic lipids ( ) containing 5 m three lipids do not undergo a phase transition in the temperature range a single transmembrane tether between lipid tails; these are synthetically of 0° to 80°C (fig. S1F). more accessible on the gram scale than fully macrocyclic archaeal lipids 25 ). Specifically, we generated three l ipids that all contained a phytanyl ( Measurement of membrane thickness by atomic acyl chain, an ether linkage to a glycerol, and a phosphocholine head force microscopy group (fig. S1A). We chose phosphocholine as a head group to increase Multilamellar vesicles were first prepared by hydration of a lipid film the probability of forming stable liposomes ( 26 ). We chose ether linkages (10 mg/ml) in Hepes buffer, followed by incubation at 50°C for to match the ether linkages that are fo und in archaea, which increase the Kim ., Sci. Adv. 2019; 5 :eaaw4783 1 May 2019 5of8 et al

6 | SCIENCE ADVANCES RESEARCH ARTICLE 30 min. Liposomal suspensions were then sonicated for 5 min and energy of CF crossing the mem- To determine the activation free liposomes was removed after 1 hour, added to a mica substrate. Excess of brane, we first needed to develop a physical model for the leakage pro- and the mica surfaces were rinsed 10 times with a 150 mM KCl solution. cess. We assumed a model where, in the initial state, all the CF is de atomic force microscope with Samples were imaged using a multimo in a solution with volume V contained in liposomes of total volume l a NanoScope IV controller (Bruker, Santa Barbara, CA) (fig. S6). The V . The passage of CF across the membrane was assumed to be a revers- s tapping mode images were acquired using silicon nitride cantilever tips ible first-order process, and the time course of CF passing through the submerged in buffer. A resonance frequency of ~8 kHz and drive am- membrane and entering the system is plitude under 100 mV were used (Asylum Research, Santa Barbara,   r depth analysis to estimate the CA).NanoScopesoftwarewasusedfo V tot kt 3 ð  exp  1 Þ % 100 ¼ð Þ C height of the lipid membranes. s V l Thermal stability of liposomes is the CF concentration in solution (normalized between 0 and C where To a glass vial, 0.2 ml of each liposome suspension (~200 nm diameter, s 100% to match the experimental data), the total volume is V V = + V , prepared in Hepes) was added into 1.8 ml of Hepes buffer. The glass vial l s tot and is the rate constant for leakage. The volume fraction of liposome k was sealed with parafilm to prevent ev aporation, and the liposome solu- V ) could not be exactly determined experimentally, but we could / V ( = 0 and 6 hours), tions were incubated at 75°C. For each time point ( t l tot assume that it was consistent between experiments and, thus, absorb 2 m l of each liposomal suspension was collected and diluted 10 times this term into the rate constant without any loss of generality. k in Hepes buffer before DLS measurement. DLS measurements re- Downloaded from To relate this rate constant to the activation free energy, we make vealed that the average diameters of these liposomes did not change use of the Eyring-Polanyi equation (Eq. 4) over the course of 6 hours at 75°C (fig. S1G), suggesting that lipo- somes composed of these synthetic lipids were thermally stable over the timeframe required for leakage experiments. ‡ ‡ T k S D D H B RT R e e ð 4 Þ k ¼ h CF leakage experiment http://advances.sciencemag.org/ The liposome stock solution obtained after gel filtration was first diluted where we have split the free energy into its enthalpic and entropic 100 times in PBS (1×, pH 7.4) to prepare 5 ml of solution A. Then, k was inserted in the kinetic equation components. This formula for solution A was diluted 10 times in PBS (1×, pH 7.4), and the resulting , and the resulting expression was used to fit the time course of for C solution (18 ml) was aliquoted into a 0.5-ml Protein LoBind Eppen- s leakage for each lipid. Using a maximum likelihood estimator, we dorf tube (0.3 ml per tube). For each temperature (22°, 37°, 50°, 60°, ‡ ‡ S D and H simultaneously fit across all temperatures to determine D and 70°C), 11 tubes corresponding to 11 time points were prepared and for each lipid. Nonlinear fitting procedures can be strongly influenced = t incubated at the corresponding temperatures. For every time point ( by outliers; however, we performed a weighted fitting procedure using : 485 nm d 8 hours), the fluorescence ( 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, an l Em the inverse variance of the data at a given time/temperature point, and : 517 nm) of one tube/temperature was measured at room tem- and l Ex this resulted in residuals that were normally distributed. All analysis perature in triplicate using a PerkinElmer EnSpire multimode plate was performed using R (www.R-project.org). on May 5, 2019 lates. Solution A was also diluted reader and Costar 96-well half-area p 10-fold in a solution of 0.5% Triton X-100 in PBS (1×, pH 7.4) to induce MD calculations of membrane properties 100% leakage. After 15 min of incubation at room temperature, the flu- Structural models for the synthetic lipids were constructed and mini- orescence of the solution was measured as above. In addition, the values mized using Maestro (Schrödinger LLC, New York, NY). The were corrected for the quenching of CF fluorescence by Triton X-100 modeled lipids all had an extended conformation along the hydro- using our plate reader (measured corr ection factor = 1.28). This leakage carbon chain. The single lipid was then translated and rotated along experiment was repeated at least three times per stock solution of lipo- X and Y directions to build an initial model membrane system com- some to ensure accuracy. prising 81 lipids. After equilibration simulations (50 ns) of this smaller ) was normalized using For each assay, the percent leakage (% leakage membrane model, the equilibrated small membrane model was repli- F Eq.1,where , represents fluorescence at represents fluorescence t F t 0 0 axes to build a bigger membrane model of 729 Y and X cated along the is the fluorescence mea- F ,and measurements at different times t triton total lipids (1458 in the case of U16). The dimensions of the bigger surement of the liposome solution including Triton X-100 membrane models were in the range of 200 × 200 × 50 Å on average   F Þ ð F 31 across the different membrane models. VMD ( ) was used to make t 0 ð 100  Þ 1 ¼ % leakage the translations and rotations of lipid in building the membrane. F ð  F Þ triton 0 Membrane simulations of 100-ns duration were performed with ), using the TIP3P explicit water model, after equilibration 32 NAMD ( Kinetic analysis at temperatures of 300, 315, and 330 K, and the pressure was main- We used Eq. 2 to determine the rates of CF leakage at different tained using the Nosé-Hoover Langevin piston method at 1 atm. The temperatures by combining individual measurements using Graph- temperature was maintained with the Nose-Hoover chain method, y coefficient of all three lipids at Pad Prism 5 software. The permeabilit and the pressure was maintained at 1 atm. The CHARMM36 lipid different temperatures was also estimated from the rates of CF leakage force field was used with a 10 Å cutoff for van der Waals with an 30 slaw(fig.S3)( ’ using Fick ) 8.5 Å switching distance and particle mesh Ewald for long-range electrostatics. Post-simulation trajectory analyses were carried out ð kt Þ 2 Þ¼ % ln ð leakage D were and D using R (www.R-project.org). The diffusion constants 2 1 Sci. Adv. 2019; 5 :eaaw4783 1 May 2019 6of8 ., Kim et al

7 | SCIENCE ADVANCES RESEARCH ARTICLE 4. E. L. Chang, Unusual thermal stability of liposomes made from bipolar tetraether lipids. determined by fitting the mean square displacement according to the , 673 Biochem. Biophys. Res. Commun. 202 – 679 (1994). 33 ) formula ( 5. M. De Rosa, A. Gambacorta, B. Nicolaus, B. Chappe, P. Albrecht, Isoprenoid ethers; backbone of complex lipids of the archaebacterium Sulfolobus solfataricus . 2 – , 249 753 Biochim. Biophys. Acta Lipids Lipid Metab. 256 (1983). tr D 4 1 2 0 6. Y. Koga, Thermal adaptation of the archaeal and bacterial lipid membranes. Archaea Þ t x 〈 ð D 4 þ 〉 ¼ t ð 5 Þ 2 2 t r þ 4 D 1 , 789652 (2012). 2012 0 7. S. F. Gilmore, A. I. Yao, Z. Tietel, T. Kind, M. T. Facciotti, A. N. Parikh, Role of squalene in the Langmuir . Halobacterium salinarum organization of monolayers derived from lipid extracts of Calculation of torsional entropy 7930 (2013). – , 7922 29 The entropy S is given by associated with torsion angle f 8. T. Koyanagi, G. Leriche, A. Yep, D. Onofrei, G. P. Holland, M. Mayer, J. Yang, Effect of i i headgroups on small-ion permeability across archaea-inspired tetraether lipid membranes. Chem. Eur. J. 22 , 8074 – 8077 (2016). 9. T. Koyanagi, G. Leriche, D. Onofrei, G. P. Holland, M. Mayer, J. Yang, Cyclohexane rings Þ d f ð r ∫ Þ ¼ Þ S 6 f ð ð R f r ln i i i i reduce membrane permeability to small ions in archaea-inspired tetraether lipids. 55 Angew. Chem. Int. Ed. , 1890 – 1893 (2016). 10. T. B. H. Schroeder, G. Leriche, T. Koyanagi, M. A. Johnson, K. N. Haengel, where is the gas constant and ( r f R ) is the probability density over f i i O. M. Eggenberger, C. L. Wang, Y. H. Kim, K. Diraviyam, D. Sept, J. Yang, M. Mayer, essentially, a normalized histogram of the — from an MD simulation + − Effects of lipid tethering in extremophile-inspired membranes on H flux at room /OH torsion. The total first-order entropy ( ) of one lipid molecule is com- 20 2440 (2016). temperature. 110 , 2430 Biophys. J. – puted as 11. E. S. Boyd, T. L. Hamilton, J. Wang, L. He, C. L. Zhang, The role of tetraether lipid 4 Front. Microbiol. composition in the adaptation of thermophilic archaea to acidity. , Downloaded from 62 (2013). N tors 12. G. D. Sprott, M. Meloche, J. C. Richards, Proportions of diether, macrocyclic diether, and 1 7 ¼ S ∑ ð Þ S i tetraether lipids in Methanococcus jannaschii grown at different temperatures. i 1 ¼ – J. Bacteriol. , 3907 3910 (1991). 173 13. Y. Matsuno, A. Sugai, H. Higashibata, W. Fukuda, K. Ueda, I. Uda, I. Sato, T. Itoh, T. Imanaka, where N is the number of torsions considered. Additional MD simula- S. Fujiwara, Effect of growth temperature and growth phase on the lipid composition tors . Thermococcus kodakaraensis of the archaeal membrane from Biosci. Biotechnol. Biochem. tions (Fig. 4) were carried out to compute these quantities in the presence http://advances.sciencemag.org/ – 108 (2009). , 104 73 and absence of CF tethered partway through one leaflet (top or upper) of 14. D. Lai, J. R. Springstead, H. G. Monbouquette, Effect of growth temperature on ether lipid membranes made of tethered and untethered lipids. Simulations were 278 (2008). biochemistry in . Archaeoglobus fulgidus Extremophiles 12 , 271 – run for T32 and U16 membrane systems, for 400 ns at a temperature 15. K. Arakawa, T. Eguchi, K. Kakinuma, Highly thermostable liposome from 72-membered macrocyclic tetraether lipid: Importance of 72-membered lipid for archaea to thrive of 300 K with and without CF ( − 2 charge state). For the simulations with 441 (2001). – 30 Chem. Lett. under hyperthermal environments. , 440 CF, the molecule was constrained to remain at the plane of the membrane 16. K. Arakawa, T. Eguchi, K. Kakinuma, 36-Membered macrocyclic diether lipid is coordinate of carbon C117 in each lipid (as shown z defined by the mean advantageous for archaea to thrive under the extreme thermal environments. plane. We xy in fig. S10), where we considered the membrane to lie in the 356 (2001). – , 347 Bull. Chem. Soc. Jpn. 74 analyzed the 26 torsional angles in th e lipid main chains (for the 26 car- Chemical Kinetics: The Study of Reaction Rates in Solution 17. K. A. Connors, (John Wiley & Sons,1990). bon atoms located in the center of the membrane for the unbranched 18. J. F. Nagle, H. L. Scott Jr., Lateral compressibility of lipid mono- and bilayers. Theory of methylene chain and phytanyl chain), 13 torsional angles in the top 513 243 (1978). – membrane permeability. Biochim. Biophys. Acta Biomembr. , 236 (where CF is located), and another 13 angles in the bottom. For the on May 5, 2019 19. J. F. Nagle, J. C. Mathai, M. L. Zeidel, S. Tristram-Nagle, Theory of passive permeability monolayer, T32, these are all in one molecule. For the bilayer-forming ,77 through lipid bilayers. J. Gen. Physiol. 131 85 (2008). – 20. B. J. Killian, J. Yundenfreund Kravitz, M. K. Gilson, Extraction of configurational entropy from lipid U16, they are in different mole cules. The T32 and U16 membranes 127 J. Chem. Phys. , 024107 (2007). molecular simulations via an expansion approximation. ,comprising64T32moleculesor were of essentially the same size 21. A. Finkelstein, A. Cass, Permeability and electrical properties of thin lipid membranes. 128 U16 molecules. 52 J. Gen. Physiol. – 172 (1968). , 145 22. J. J. Hoyt, M. Asta, B. Sadigh, Test of the universal scaling law for the diffusion coefficient in liquid metals. 597 (2000). – , 594 85 Phys. Rev. Lett. SUPPLEMENTARY MATERIALS 23. I. Yokoyama, S. Tsuchiya, Excess entropy, diffusion coefficient, viscosity coefficient and Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ , surface tension of liquid simple metals from diffraction data. 43 Mater. Trans. content/full/5/5/eaaw4783/DC1 72 (2002). 67 – Fig. S1. Synthesis and characterization of archaea-inspired lipids. 24. M. Dzugutov, A universal scaling law for atomic diffusion in condensed matter. Nature Fig. S2. Liposome radius distribution. – 381 139 (1996). , 137 Fig. S3. Time dependence of CF leakage at different temperatures for ~80-nm liposomes. 25. T. Eguchi, K. Ibaragi, K. Kakinuma, Total synthesis of archaeal 72-membered macrocyclic Fig. S4. Temperature-dependent CF release profile from liposomes ~200 nm in diameter. tetraether J. Org. Chem. 2698 (1998). 63 , 2689 – lipids. Fig. S5. Cross-sectional views of simulated membranes. 26. S. Ohki, Cell and Model Membrane Interactions (Plenum Press, 1991). Fig. S6. 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8 | RESEARCH ARTICLE SCIENCE ADVANCES Institute of General Medical Sciences, NIH (grant GM061300 to M.K.G.). The contents of this 32. J. C. Phillips, R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, C. Chipot, R. D. Skeel, publication are solely the responsibility of the authors and do not necessarily represent the J. Comput. Chem. 26 , L. Kalé, K. Schulten, Scalable molecular dynamics with NAMD. official views of the NIH. Y.H.K., G.L., T.K., A.G., and J.Y. planned the study, Author contributions: – 1802 (2005). 1781 33. J. Wohlert, O. Edholm, Dynamics in atomistic simulations of phospholipid membranes: designed the leakage experiments, and analyzed the results. D.O. and G.P.H. conducted DSC , 125 J. Chem. Phys. Nuclear magnetic resonance relaxation rates and lateral diffusion. measurements. J.P. and N.G. conducted cryo-EM experiments. K.D., K.G., M.K.G., and D.S. designed 204703 (2006). the computational study and analyzed the results. G.L. M.K.G., M.M., D.S., and J.Y. wrote the 34. D. A. Case, V. Babin, J.T. Berryman, R. M. Betz, Q. Cai, D. S. Cerutti, T. E. Cheatham III, manuscript. M.K.G. has an equity interest in, and is a cofounder and scientific Competing interests: T. A. Darden, R. E. Duke, H. Gohlke, A. W. Goetz, S. Gusarov, N. Homeyer, P. Janowski, advisor of, VeraChem LLC. All other authors declare that they have no competing financial and J. Kaus, I. Kolossváry, A. Kovalenko, T. S. Lee, S. LeGrand, T. Luchko, R. Luo, B. Madej, K. Merz, All data needed to evaluate the conclusions Dataand materials availability: nonfinancial interests. F. Paesani, D. R. Roe, A. Roitberg, C. Sagui, R. Salomon-Ferrer, Gustavo de Miranda Seabra, in the paper are present in the paper and/or the Supplementary Materials. Additional data C. Simmerling, W. Smith, J. M. Swails, R.C. Walker, J. Wang, R.M. Wolf, X. Wu, P.A. Kollman, related to this paper may be requested from the authors. AMBER 14 (University of California, San Francisco, 2014). 35. J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollman, D. A. Case, Development and testing of a Submitted 23 December 2018 – , 1157 25 J. Comput. Chem. general amber force field. 1174 (2004). Accepted 14 March 2019 36. C. J. Dickson, B. D. Madej, Å. A. Skjevik, R. M. Betz, K. Teigen, I. R. Gould, R. C. Walker, Published 1 May 2019 – 879 (2014). Lipid14: The amber lipid force field. 10 J. Chem. Theory Comput. , 865 10.1126/sciadv.aaw4783 Acknowledgments Citation: Y. H. Kim, G. Leriche, K. Diraviyam, T. Koyanagi, K. Gao, D. Onofrei, J. Patterson, Funding: We acknowledge the financial support from the Air Force Office of Scientific Research A.Guha,N.Gianneschi,G.P.Holland,M.K.Gilson,M.Mayer,D.Sept,J.Yang,Entropic (FA9550-12-1-0435 and FA9550-17-1-0282). This work was also supported, in part, by the National , eaaw4783 (2019). Sci. Adv. effects enable life at extreme temperatures. 5 Downloaded from http://advances.sciencemag.org/ on May 5, 2019 et al ., Kim 2019; 5 :eaaw4783 1 May 2019 8of8 Sci. Adv.

9 Entropic effects enable life at extreme temperatures Young Hun Kim, Geoffray Leriche, Karthik Diraviyam, Takaoki Koyanagi, Kaifu Gao, David Onofrei, Joseph Patterson, Anirvan Guha, Nathan Gianneschi, Gregory P. Holland, Michael K. Gilson, Michael Mayer, David Sept and Jerry Yang (5), eaaw4783. 5 Sci Adv DOI: 10.1126/sciadv.aaw4783 Downloaded from ARTICLE TOOLS http://advances.sciencemag.org/content/5/5/eaaw4783 SUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2019/04/29/5.5.eaaw4783.DC1 MATERIALS http://advances.sciencemag.org/ REFERENCES This article cites 33 articles, 2 of which you can access for free http://advances.sciencemag.org/content/5/5/eaaw4783#BIBL PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions on May 5, 2019 Use of this article is subject to the Terms of Service (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 New Science Advances York Avenue NW, Washington, DC 20005. 2017 © The Authors, some rights reserved; exclusive licensee American Science Advances is a Association for the Advancement of Science. No claim to original U.S. Government Works. The title registered trademark of AAAS.

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