Determining Cholesterol Binding to Membrane Proteins by Cholesterol ¹³C Labeling in Yeast and Dynamic Nuclear Polarization NMR.

ABSTRACT: We present a general strategy for determining the cholesterol-binding site of eukaryotic membrane proteins in native-like lipid membranes by NMR spectroscopy. The strategy combines yeast biosynthetic ¹³C enrichment of cholesterol with detection of protein-cholesterol ¹³C-¹³C cross peaks in 2D correlation NMR spectra under the dynamic nuclear polarization (DNP) condition. Low-temperature DNP not only allows high-sensitivity detection of weak protein-cholesterol cross peaks in 2D spectra but also immobilizes cholesterol and protein to enable intermolecular distance measurements. We demonstrate this approach on the influenza M2 protein, which utilizes cholesterol to conduct membrane scission in the last step of virus budding and release from the host cell plas

SUBMITTER: Elkins MR 

PROVIDER: S-EPMC6361393 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature