Lipid Ordering

Lipid ordering refers to the capacity of certain lipids — primarily polycyclic isoprenoids such as sterols and Hopanoids — to constrain the rotational freedom and lateral packing of lipids within a bilayer. This ordering is the physical basis for the liquid-ordered (Lo) phase, a thermodynamic intermediate between the fluid liquid-disordered (Ld) phase and the rigid crystalline gel phase.

The Liquid-Ordered Phase

The Lo phase is characterized by:

• Tightly packed, conformationally restricted acyl chains (ordered)
• Lateral diffusivity that remains fluid (not crystalline)
• Reduced diffusion relative to Ld (~7-fold slower, based on FCS measurements of ~14 ms vs. ~2 ms diffusion times)

In eukaryotes, cholesterol interacts with sphingolipids to produce Lo phases, providing the mechanistic basis for membrane lateral segregation, lipid raft formation, and a fluid but mechanically robust plasma membrane. Until our work, this capacity was thought to be unique to sterol-containing organisms.

Hopanoid-Based Ordering

We demonstrated that Diplopterol shares all essential ordering properties of cholesterol in model membranes (Sáenz et al., 2012, PNAS):

• Inhibition of gel phase formation in sphingomyelin
• Ordering of saturated acyl chains (comparable C-laurdan GP values)
• Formation of coexisting Lo and Ld phases in GUVs
• Condensing and ordering of lipid A

In living bacteria, we confirmed that hopanoids determine outer membrane order in [[methylobacterium-extorquens|Methylobacterium extorquens]], interacting with lipid A in a manner analogous to cholesterol-sphingolipid interactions in eukaryotes (Sáenz et al., 2015, PNAS).

Measuring Membrane Order

Several experimental approaches quantify membrane order:

• C-laurdan spectroscopy: the generalized polarization (GP) index reports on membrane hydration, which correlates with lipid packing order. Higher GP = more ordered.
• Di-4-ANEPPDHQ: another order-sensitive fluorescent probe used to confirm C-laurdan results.
• Langmuir monolayers: pressure–area isotherms reveal gel-liquid transitions and condensation effects. The percent condensation quantifies how much a polycyclic isoprenoid reduces the area per molecule.
• FCS (fluorescence correlation spectroscopy): measures lateral diffusion times of labeled lipids, distinguishing Lo (slow) from Ld (fast) domains.
• Detergent resistance: association with detergent-resistant membrane fractions (DRMs) indicates partitioning into ordered domains.

Functional Consequences

Membrane order is directly linked to:

• Barrier function: ordered membranes have reduced permeability
• Protein function: energy-dependent multidrug efflux is impaired when hopanoid-mediated order is lost in M. extorquens
• Lateral segregation: ordered and disordered phases can coexist and segregate, creating functional membrane domains
• pH buffering: hopanoids moderate pH-induced changes in lipid A order, potentially protecting bacteria in acidic environments

Evolutionary Implications

Because hopanoid synthesis does not require molecular oxygen, the capacity for membrane ordering could predate the oxygenation of Earth’s surface. Our findings suggest that the evolution of isoprenoidal cyclase enzymes may have been a pivotal event in biological complexity — enabling a second biochemically active liquid membrane phase and the decoupling of lipid diffusivity from acyl chain conformational freedom.