bi5b00127_si_001.pdf (329.66 kB)

Investigation of the Curvature Induction and Membrane Localization of the Influenza Virus M2 Protein Using Static and Off-Magic-Angle Spinning Solid-State Nuclear Magnetic Resonance of Oriented Bicelles

Download (329.66 kB)
journal contribution
posted on 07.04.2015 by Tuo Wang, Mei Hong
A wide variety of membrane proteins induce membrane curvature for function; thus, it is important to develop new methods to simultaneously determine membrane curvature and protein binding sites in membranes with multiple curvatures. We introduce solid-state nuclear magnetic resonance (NMR) methods based on magnetically oriented bicelles and off-magic-angle spinning (OMAS) to measure membrane curvature and the binding site of proteins in mixed-curvature membranes. We demonstrate these methods on the influenza virus M2 protein, which not only acts as a proton channel but also mediates virus assembly and membrane scission. An M2 peptide encompassing the transmembrane (TM) domain and an amphipathic helix, M2(21–61), was studied and compared with the TM peptide (M2TM). Static 31P NMR spectra of magnetically oriented 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles exhibit a temperature-independent isotropic chemical shift in the presence of M2(21–61) but not M2TM, indicating that the amphipathic helix confers the ability to generate a high-curvature phase. Two-dimensional (2D) 31P spectra indicate that this high-curvature phase is associated with the DHPC bicelle edges, suggestive of the structure of budding viruses from the host cell. 31P- and 13C-detected 1H relaxation times of the lipids indicate that the majority of M2(21–61) is bound to the high-curvature phase. Using OMAS experiments, we resolved the 31P signals of lipids with identical headgroups based on their distinct chemical shift anisotropies. On the basis of this resolution, 2D 1H–31P correlation spectra show that the amide protons in M2(21–61) correlate with the DMPC but not DHPC 31P signal of the bicelle, indicating that a small percentage of M2(21–61) partitions into the planar region of the bicelles. These results show that the amphipathic helix induces high membrane curvature and localizes the protein to this phase, in good agreement with the membrane scission function of the protein. These bicelle-based relaxation and OMAS solid-state NMR techniques are generally applicable to curvature-inducing membrane proteins such as those involved in membrane trafficking, membrane fusion, and cell division.

History

Exports