Dataset Information

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 plasma membrane. A 13C-13C double-quantum filter was employed to significantly simplify the 2D 13C-13C correlation spectra and facilitate the identification of protein-cholesterol cross peaks. A number of cross peaks between the M2 transmembrane residues' side chains and the cholesterol sterol group were detected, which complement recently measured protein contacts to the isooctyl tail of cholesterol to define an extended binding interface. These results provide atomic-level evidence of M2-cholesterol interaction to cause membrane curvature and scission, and the approach is generally applicable to other eukaryotic membrane proteins for understanding the influence of cholesterol on membrane protein function.

SUBMITTER: Elkins MR

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

REPOSITORIES: biostudies-literature

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

Elkins Matthew R MR Sergeyev Ivan V IV Hong Mei M

Journal of the American Chemical Society 20181030 45

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 cr ...[more]

PMID: 30338997

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Project description:Isotopic enrichment of 29Si and DNP-enhanced NMR spectroscopy are combined to determine the detailed surface structure of a silicated alumina catalyst. The significant sensitivity enhancement provided by DNP is vital to the acquisition of multinuclear and multidimensional experiments that provide information on the atomic-level structure of the species present at the surface. Isotopic enrichment not only facilitates spectral acquisition, particularly given the low (1.5 wt %) Si loading, but also enables spectra with higher resolution than those acquired using DNP to be obtained. The unexpected similarity of conventional, CP, and DNP NMR spectra is attributed to the presence of adventitious surface water that forms a sufficiently dense 1H network at the silica surface so as to mediate efficient polarization transfer to all Si species regardless of their chemical nature. Spectra reveal the presence of Si-O-Si linkages at the surface (identified as Q4(3Al)-Q4(3Al)) and confirm that the anchoring of the surface overlayer with the alumina occurs through AlIV and AlV species only. This suggests the presence of Q3/Q4 Si at the surface affects the neighboring Al species, modifying the surface structure and making it less likely AlVI environments are in close spatial proximity. In contrast, Q1/Q2 species, bonded to the surface by fewer covalent bonds, have less of an effect on the surface, and more AlVI species are consequently found nearby. The combination of isotropic enrichment and DNP provides a definitive and fully quantitative description of the Si-modified alumina surface, and we demonstrate that almost one-third of the silicon at the surface is connected to another Si species, even at the low level of coverage used, lowering the propensity for the formation of Brønsted acid sites. This suggests that a variation in the synthetic procedure might be required to obtain a more even coverage for optimum performance. The work here will allow for more rigorous future investigations of structure-function relationships in these complex materials.

Dataset Information

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

Publications

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

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