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 C enrichment in combination with magic-angle spinning (MAS) methods. We quantify the incorporation levels and demonstrate high sensitivity and resolution in 2DC-C and H-C 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.

Amphotericin B (AmB) is the archetype for small molecules that form ion channels in living systems, and has recently been shown to replace a missing protein ion transporter and thereby restore physiology in yeast. Molecular modeling studies predict that AmB self-assembles in lipid membranes with the polyol region lining a channel interior that funnels to its narrowest region at the C3-hydroxyl group. This model predicts that modification of this functional group would alter conductance of the AmB ion channel. To test this hypothesis, the C3-hydroxyl group was synthetically deleted and the resulting derivative, C3deoxyAmB (C3deOAmB), was characterized using multidimensional NMR experiments and single ion channel electrophysiology recordings. C3deOAmB possesses the same macrocycle conformation as AmB and retains the capacity to form transmembrane ion channels, yet the conductance of the C3deOAmB channels is threefold lower than that of AmB channels. Thus, the C3-hydroxyl group plays an important role in AmB ion channel conductance, and synthetic modifications at this position may provide an opportunity for further tuning of channel functions.

Sterols, vital and abundant components of plasma membranes, are involved in numerous cellular biochemical processes such as regulation of membrane properties and signaling events. However, these sterol interactions are not completely elucidated at the atomic level; this impedes further investigations into disorders related to these biological activities. Combining solid-state NMR (SSNMR), high yield biosynthesis of isotopically labeled sterols, and molecular dynamics, we aim to understand the key interactions that determine the sterol-specificity to amphotericin B (AmB), a powerful but toxic antifungal drug used to treat life-threatening fungal infections. AmB forms large aggregates that are neither crystalline nor soluble, making structural information difficult or impossible to obtain via x-ray crystallography or solution-state NMR. We have recently determined that AmB kills yeast cells primarily by binding to ergosterol 1 (Erg) and forming a large extramembranous sterol sponge. 2 These findings were enabled by the biosynthesis of fractionally 13 C labeled Erg and state-of-the-art SSNMR techniques. The sterol sponge model hypothesizes that interactions of AmB with Erg determine its ability to kill yeast, whereas binding to cholesterol is responsible for determining its toxicity in human cells. SSNMR spectroscopy is uniquely able to detect and quantify the binding of sterols to AmB in the sterol sponge in atomistic detail. These studies provide a roadmap towards an improved therapeutic index 3 for this drug while simultaneously elucidating additional details about the molecular mechanism of AmB-sterol interactions.(1)''Amphotericin primarily kills yeast by simply binding ergosterol'', Gray et al.