Diplopterol

Diplopterol is the simplest bacterial hopanoid, consisting of the pentacyclic hopane ring system with a single hydroxyl group at C-22. It is the primary hopanoid in several bacteria, including [[methylobacterium-extorquens|Methylobacterium extorquens]], where it and its C-2 methylated derivative (2-methyl-diplopterol) together comprise ~19 mol% of total outer membrane lipids (Sáenz et al., 2015, PNAS).

Membrane-Ordering Properties

We established that diplopterol shares the key lipid-ordering properties of cholesterol through a series of biophysical experiments (Sáenz et al., 2012, PNAS):

• Gel phase inhibition: Diplopterol eliminates the gel-liquid phase transition in sphingomyelin monolayers, as cholesterol does.
• Acyl chain ordering: Diplopterol orders sphingomyelin comparably to cholesterol, as measured by C-laurdan GP spectroscopy.
• Lo phase formation: In GUVs containing SM/DOPC/diplopterol (1:1:0.75), diplopterol induces liquid-liquid phase separation into Lo and Ld domains with order and diffusivity essentially identical to cholesterol-containing GUVs (~14 ms diffusion time for Atto532-SM vs. ~2 ms in DOPC).
• Lipid A ordering: Both diplopterol and cholesterol condense lipid A, inhibit pH-induced gel transitions, and buffer pH-induced changes in lipid A order.

Selectivity for Saturated Lipids

A critical difference from cholesterol: diplopterol interacts favorably only with saturated lipids (lipid A, sphingomyelin) and repulsively with unsaturated phospholipids, as measured by Gibbs excess free energy of mixing (ΔGex) on a Langmuir trough. Cholesterol, by contrast, interacts favorably with phospholipids across a range of unsaturation. This selectivity is consistent with the lipid composition of the bacterial outer membrane, where hopanoids encounter saturated lipid A in the outer leaflet (Sáenz et al., 2015, PNAS).

In Vivo Function

In M. extorquens, diplopterol determines outer membrane order. Genetic deletion of squalene-hopene cyclase (ΔSHC) reduces OM order; loading diplopterol into ΔSHC membranes using methyl-β-cyclodextrin restores it. Remarkably, loading cholesterol into ΔSHC membranes also restores order, demonstrating functional interchangeability between these structurally distinct polycyclic isoprenoids.

Later work revealed that diplopterol’s ordering capacity depends on the position of the phospholipid double bond: diplopterol condenses Δ11-unsaturated lipids effectively but has an unfavorable interaction with the common Δ9 position, a constraint traced to steric overlap between the hopanoid’s methyl groups and the double bond (Nguyen et al., 2024, Biophysical Journal).