(2)H,(13)C-Cholesterol for Dynamics and Structural Studies of Biological Membranes

[Image: see text] We present a cost-effective means of (2)H and (13)C enrichment of cholesterol. This method exploits the metabolism of (2)H,(13)C-acetate into acetyl-CoA, the first substrate in the mevalonate pathway. We show that growing the cholesterol producing strain RH6827 of Saccharomyces cerevisiae in (2)H,(13)C-acetate-enriched minimal media produces a skip-labeled pattern of deuteration. We characterize this cholesterol labeling pattern by mass spectrometry and solid-state nuclear magnetic resonance spectroscopy. It is confirmed that most (2)H nuclei retain their original (2)H–(13)C bonds from acetate throughout the biosynthetic pathway. We then quantify the changes in (13)C chemical shifts brought by deuteration and the impact upon (13)C–(13)C spin diffusion. Finally, using adiabatic rotor echo short pulse irradiation cross-polarization ((RESPIRATION)CP), we acquire the (2)H–(13)C correlation spectra to site specifically quantify cholesterol dynamics in two model membranes as a function of temperature. These measurements show that cholesterol acyl chains at physiological temperatures in mixtures of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin, and cholesterol are more dynamic than cholesterol in POPC. However, this overall change in motion is not uniform across the cholesterol molecule. This result establishes that this cholesterol labeling pattern will have great utility in reporting on cholesterol dynamics and orientation in a variety of environments and with different membrane bilayer components, as well as monitoring the mevalonate pathway product interactions within the bilayer. Finally, the flexibility and universality of acetate labeling will allow this technique to be widely applied to a large range of lipids and other natural products.