1H-detected fast magic-angle-spinning (MAS) solid-state NMR is emerging as an important analysis method for proteins of which only sub-milligram amounts are available. This makes also integral membrane proteins which can be expressed at low yields only accessible for a structural investigation by solid-state NMR.
On the example of the Hepatitis C viral membrane protein NS4B (Non-Structural membrane protein 4B), which is predicted to be an α-helical integral membrane protein, we have tested the spectral quality of preparations reconstituted into liposomes of various lipid composition. We find a strong dependence of the spectral resolution on the lipid composition that seems to be connected to the lipid phase transition temperature, with spectra recorded above the lipid phase transition temperature yielding high resolution and those recorded below the lipid phase transition temperature showing broad signals. However, for all lipids investigated, the bulk 1H and 15N transverse relaxation rate constants are very similar, pointing to inhomogeneous line broadening as the determining factor for spectral resolution.
Nevertheless, the short transverse relaxation times of NS4B (bulk 1H R2’ rate constants ~ 5 ms) put substantial challenges for higher-dimensional spectral backbone assignment experiments. We employ CP (cross-polarization) and DREAM (dipolar recoupling enhanced by amplitude modulation) for coherence transfer. Based on the 1H, 15N and 13C relaxation rate constants which are effective during the different evolution times and coherence-transfer periods, we have optimized HN detected three-dimensional backbone assignment experiments. The high MAS frequencies of 100 kHz needed for resolution require relatively high RF amplitudes of the inverse nuclei during the CP or DREAM mixing periods, leading to non-selective transfers. We have identified several unwanted magnetization-loss mechanisms and have optimized the respective backbone assignment experiments accordingly.