Spectroscopic assignment of NMR spectra for oriented uniformly labeled membrane proteins embedded in their native-like bilayer environment is essential for their structure determination. However, sequence-specific assignment in oriented-sample (OS) NMR is often complicated by insufficient resolution and spectral crowding. Therefore, the assignment process is usually done by a laborious and expensive "shotgun" method involving multiple selective labeling of amino acid residues. Presented here is a strategy to overcome poor spectral resolution in crowded regions of 2D spectra by selecting resolved "seed" residues via soft Gaussian pulses inserted into spin-exchange separated local-field experiments. The Gaussian pulse places the selected polarization along the z-axis while dephasing the other signals before the evolution of the H-N dipolar couplings. The transfer of magnetization is accomplished via mismatched Hartmann-Hahn conditions to the nearest-neighbor peaks via the proton bath. By optimizing the length and amplitude of the Gaussian pulse, one can also achieve a phase inversion of the closest peaks, thus providing an additional phase contrast. From the superposition of the selective spin-exchanged SAMPI4 onto the fully excited SAMPI4 spectrum, the N sites that are directly adjacent to the selectively excited residues can be easily identified, thereby providing a straightforward method for initiating the assignment process in oriented membrane proteins.
Multidimensional separated local-field and spin-exchange experiments employed by oriented-sample solid-state NMR are essential for structure determination and spectroscopic assignment of membrane proteins reconstituted in macroscopically aligned lipid bilayers. However, these experiments typically require a large number of scans in order to establish interspin correlations. Here we have shown that a combination of optimized repetitive cross polarization (REP-CP) and membrane-embedded free radicals allows one to enhance the signal-to-noise ratio by factors 2.4-3.0 in the case of Pf1 coat protein reconstituted in magnetically aligned bicelles with their normals being either parallel or perpendicular to the main magnetic field. Notably, spectral resolution is not affected at the 2:1 radical-to-protein ratio. Spectroscopic assignment of Pf1 coat protein in the parallel bicelles has been established as an illustration of the method. The proposed methodology will advance applications of oriented-sample NMR technique when applied to samples containing smaller quantities of proteins and three-dimensional experiments.
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