H/C/N triple-resonance experiments for structure determinaton of membrane proteins by oriented-sample NMR.

The benefits of triple-resonance experiments for structure determination of macroscopically oriented membrane proteins by solid-state NMR are discussed. While double-resonance H/N experiments are effective for structure elucidation of alpha-helical domains, extension of the method of oriented samples to more complex topologies and assessing side-chain conformations necessitates further development of triple-resonance (H/C/N) NMR pulse sequences. Incorporating additional spectroscopic dimensions involving C spin-bearing nuclei, however, introduces essential complications arising from the wide frequency range of the H-C dipolar couplings and C CSA (>20 ​kHz), and the presence of the C-C homonuclear dipole-dipole interactions. The recently reported ROULETTE-CAHA pulse sequence, in combination with the selective z-filtering, can be used to evolve the structurally informative H-C dipolar coupling arising from the aliphatic carbons while suppressing the signals from the carbonyl and methyl regions. Proton-mediated magnetization transfer under mismatched Hartman-Hahn conditions (MMHH) can be used to correlate C and N nuclei in such triple-resonance experiments for the subsequent N detection. The recently developed pulse sequences are illustrated for n-acetyl Leucine (NAL) single crystal and doubly labeled Pf1 coat protein reconstituted in magnetically aligned bicelles. An interesting observation is that in the case of N-labeled NAL measured at C natural abundance, the triple (H/C/N) MMHH scheme predominantly gives rise to long-range intermolecular magnetization transfers from C to N spins; whereas direct Hartmann-Hahn C/N transfer is entirely intramolecular. The presented developments advance NMR of oriented samples for structure determination of membrane proteins and liquid crystals.