Proton-evolved local-field solid-state NMR studies of cytochrome b5 embedded in bicelles, revealing both structural and dynamical information.

Structural biology of membrane proteins has rapidly evolved into a new frontier of science. Although solving the structure of a membrane protein with atomic-level resolution is still a major challenge, separated local field (SLF) NMR spectroscopy has become an invaluable tool in obtaining structural images of membrane proteins under physiological conditions. Recent studies have demonstrated the use of rotating-frame SLF techniques to accurately measure strong heteronuclear dipolar couplings between directly bonded nuclei. However, in these experiments, all weak dipolar couplings are suppressed. On the other hand, weak heteronuclear dipolar couplings can be measured using laboratory-frame SLF experiments, but only at the expense of spectral resolution for strongly dipolar coupled spins. In the present study, we implemented two-dimensional proton-evolved local-field (2D PELF) pulse sequences using either composite zero cross-polarization (COMPOZER-CP) or windowless isotropic mixing (WIM) for magnetization transfer. These PELF sequences can be used for the measurement of a broad range of heteronuclear dipolar couplings, allowing for a complete mapping of protein dynamics in a lipid bilayer environment. Experimental results from magnetically aligned bicelles containing uniformly (15)N-labeled cytochrome b(5) are presented and theoretical analyses of the new PELF sequences are reported. Our results suggest that the PELF-based experimental approaches will have a profound impact on solid-state NMR spectroscopy of membrane proteins and other membrane-associated molecules in magnetically aligned bicelles.