Filling the Gap with Long n -Alkanes: Incorporation of C20 and C30 into Phospholipid Membranes.

Investigating how hydrophobic molecules mix with phospholipid bilayers and how they affect membrane properties is commonplace in biophysics. Despite this, a molecular-level empirical description of a membrane model as simple as a phospholipid bilayer with long linear hydrophobic chains incorporated is still missing. Here, we present an unprecedented molecular characterization of the incorporation of two long n -alkanes, n -eicosane (C20) and n -triacontane (C30) with 20 and 30 carbons, respectively, in phosphatidylcholine (PC) bilayers using a combination of experimental techniques ( 2 H NMR, 31 P NMR, 1 H- 13 C dipolar recoupling solid-state NMR, X-ray scattering, and cryogenic electron microscopy) and atomistic molecular dynamics (MD) simulations. At low hydration, deuterated C20 and C30 yield 2 H NMR spectra evidencing anisotropic-motion, which demonstrates their miscibility in PC membranes up to a critical alkane-to-acyl-chain volume fraction, ϕ c . The acquired 2 H NMR spectra of C20 and C30 have notably different lineshapes. At low alkane volume fractions below ϕ c , CHARMM36 MD simulations predict such 2 H NMR spectra qualitatively and thus enable an atomistic-level interpretation of the spectra. Above ϕ c , the 2 H NMR lineshapes become characteristic of motions in the intermediate-regime that, together with the MD simulation results, suggest the onset of immiscibility between the alkane molecules and the acyl chains. For all the systems investigated, the phospholipid molecular structure is unperturbed by the presence of the alkanes. However, at conditions of excess hydration and at surprisingly low alkane fractions below ϕ c , a peak characteristic of isotropic motion is observed in both the 2 H spectra of the alkanes and 31 P spectra of the phospholipids, strongly indicating that the incorporation of the alkanes induces a reduction on the average radius of the lipid vesicles.