New Solid-State NMR Methodologies for Structure Determination of Membrane Proteins: Sensitivity Enhancement, Spectroscopic Assignment, and Macroscopic Alignment

Oriented-sample NMR (OS NMR) has emerged as a powerful tool for structure determination of membrane proteins in their native lipid environment. In the present work, we address three issues of critical importance for OS NMR of membrane proteins: sensitivity enhancement, spectroscopic assignment, and macroscopic alignment. (i). A repetitive-contact cross-polarization (REP-CP) scheme was developed that allows one to fully transfer the thermodynamic limit of polarization from the abundant protons to low-gamma nuclei. As a result, a factor of two (2) enhancement in sensitivity was gained over the single-contact traditional CP, whereas 45% average intensity gain is achieved as compared to CP-MOIST, currently the most widely used CP scheme in OS NMR. (ii). We have also developed a strategy for assigning solid-state NMR spectra of Pf1 coat protein reconstituted in magnetically aligned bicelles, and obtained a two-dimensional spin-exchanged version of the SAMPI4 spectrum correlating 15N chemical shift and 15N-1H dipolar couplings. Combining the spin-exchanged version with the original SAMPI4 experiment makes it possible to establish sequence-specific assignments, and this technique is generally applicable to other membrane proteins. Notably, only a single uniformly labeled protein sample is required as opposed to multiple selectively labeled samples currently employed for spectroscopic assignment. Using sensitivity enhancement techniques such as REP-CP assists in further elucidating cross peaks and allows the establishment of correlations between the adjacent residue along the backbone. Simulation accurately predicts the optimal MMHH condition as well as explains experimental trends. (iii). Finally, we also successfully applied a new method to align membrane proteins into the lipid bilayers and obtained high-resolution OS NMR spectra of the uniformly 15N labeled transmembrane domain of Pf1 coat protein. Nanoporous anodic aluminum oxide (AAO) sheets have been proven to be an alternative alignment system for the study of membrane proteins in their native environments by means of solid-state NMR. AAO-supported bilayers have the potential of providing an alternative membrane mimic of highly flexible composition for the structure-function studies of membrane proteins.