Investigating the Biophysical Basis of Mosaic Spread in Aligned Samples

Abstract

Oriented solid-state NMR (O-SSNMR) is a powerful technique to probe the structure and dynamics of membrane proteins. One of the most popular experiments on aligned samples is PISEMA (polarization inversion spin exchange at the magic angle), which is most often used to correlate the 1H-15N dipolar coupling with the 15N anisotropic chemical shift in membrane proteins. The spectral patterns in PISEMA are directly sensitive to both the secondary structure geometry with respect to the lipid bilayer normal and the mosaic spread within aligned samples. The latter will result in an increased linewidth of the peaks. Using the drug transporter EmrE as a model protein we investigated conditions that can reduce the mosaic spread in aligned samples and therefore increase the spectral resolution. EmrE is an integral membrane protein with four transmembrane domains and 110 residues that forms an anti-parallel and asymmetric homodimer. Interestingly, we discovered that ether linked lipids as well as the presence of conservative mutations in EmrE can profoundly increase the spectral resolution and therefore sensitivity of the PISEMA experiment, which implies a direct reduction in the mosaic spread of the samples for O-SSNMR. These results improve the feasibility of employing aligned methodology to determine the tilt angles and probe the conformational dynamics of membrane proteins with respect to the lipid bilayer.