Functional convergence of hopanoids and sterols in membrane ordering

Sáenz, J. P.; Sezgin, E.; Schwille, P.; Simons, K. (2012) Proceedings of the National Academy of Sciences, 109(35), 14236–14240. DOI: 10.1073/pnas.1212141109

Summary

Liquid-ordered (Lo) phases are biochemically active membrane states that had been considered a unique consequence of sterol-lipid interactions in eukaryotes. We showed that Diplopterol, the simplest bacterial hopanoid, shares the capacity of cholesterol to order saturated lipids and to form a Lo phase in model membranes. Using Langmuir monolayers, C-laurdan spectroscopy, giant unilamellar vesicles (GUVs), and fluorescence correlation spectroscopy, we demonstrated that diplopterol inhibits gel phase formation in sphingomyelin, orders saturated acyl chains comparably to cholesterol, and induces liquid-liquid phase separation. Because hopanoid biosynthesis does not require molecular oxygen, these results suggest that ordered, biochemically active membranes could have evolved before the oxygenation of Earth’s surface.

Key Findings

• Diplopterol inhibits gel phase formation in sphingomyelin monolayers, the same effect exerted by cholesterol, as measured by Langmuir trough isotherms at 25 °C.
• The ordering effect of diplopterol on sphingomyelin liposomes (measured by C-laurdan generalized polarization) is comparable to that of cholesterol at 2:1 SM:lipid molar ratios.
• In GUVs composed of SM/DOPC with either cholesterol or diplopterol (1:1:0.75), both lipids induce liquid-liquid phase separation into coexisting Lo and Ld domains with essentially identical order (GP values) and diffusivity (~14 ms diffusion time for Atto532-SM, compared to ~2 ms in DOPC GUVs).
• Diplopterol exhibits a weak ordering effect on unsaturated phospholipids, in contrast to cholesterol — a key difference explored further in later work (Sáenz et al., 2015, PNAS).
• Both diplopterol and cholesterol condense lipid A (kdo-lipid A) in monolayers, inhibit pH-induced gel phase transitions in lipid A, and buffer pH-induced changes in membrane order — suggesting a physiologically relevant role in the bacterial outer membrane.
• The ordering effect of diplopterol was detectable at molar concentrations as low as 5%, within the physiological range of hopanoid abundance in bacteria.

Methods

• Langmuir monolayers: pressure–area isotherms on a teflon trough with Wilhelmy plate to measure condensation effects and phase transitions.
• C-laurdan spectroscopy: generalized polarization (GP) measurements on liposomes at 50 °C (above SM gel-liquid transition) and 25 °C for lipid A experiments.
• GUV microscopy: two-photon confocal fluorescence microscopy of electroswelled GUVs stained with C-laurdan to visualize Lo/Ld phase coexistence.
• Fluorescence correlation spectroscopy (FCS): diffusion time measurements of Atto532-labeled sphingomyelin in GUVs to quantify membrane fluidity differences between Lo and Ld phases.
• Experiments performed at MPI-CBG, Dresden, supported by Alexander von Humboldt Foundation and NSF IRFP.

Significance

This paper established the fundamental biophysical equivalence of Hopanoids and sterols in membrane ordering — the property that gives rise to Lo phases and membrane lateral organization. Prior to this work, hopanoids had been called “sterol surrogates” based on structural similarity and condensing effects, but the ability to form a Lo phase had not been demonstrated. The evolutionary implication is substantial: because hopanoid synthesis is anaerobic while sterol synthesis requires O₂, ordered membranes and their functional consequences (lateral segregation, domain formation) could predate the Great Oxidation Event by over a billion years.