Solid-State NMR of highly ¹³C-enriched cholesterol in lipid bilayers

Cholesterol (Chol) is vital for cell function as it is essential to a myriad of biochemical and biophysical processes. The atomistic details of Chol's interactions with phospholipids and proteins is therefore of fundamental interest, and NMR offers unique opportunities to interrogate these properties at high resolution. Towards this end, here we describe approaches for examining the structure and dynamics of Chol in lipid bilayers using high levels of ¹³C enrichment in combination with magic-angle spinning (MAS) methods. We quantify the incorporation levels and demonstrate high sensitivity and resolution in 2D ¹³C-¹³C and ¹H-¹³C spectra, enabling de novo assignments and site-resolved order parameter measurements obtained in a fraction of the time required for experiments with natural abundance sterols. We envision many potential future applications of these methods to study sterol interactions with drugs, lipids and proteins.