¹H/¹³C/¹⁵N 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-1/N-15 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-1/C-13/N-15) NMR pulse sequences. Incorporating additional spectroscopic dimensions involving C-13 spin-bearing nuclei, however, introduces essential complications arising from the wide frequency range of the H-1 -C-13 dipolar couplings and C-13 CSA (>20 kHz), and the presence of the C-13 -C-13 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-1-C-13 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-13 and N-15 nuclei in such triple-resonance experiments for the subsequent N-15 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-15-labeled NAL measured at C-13 natural abundance, the triple (H-1/C-13/N-15) MMHH scheme predominantly gives rise to long-range intermolecular magnetization transfers from C-13 to N-15 spins; whereas direct Hartmann-Hahn C-13/N-15 transfer is entirely intramolecular. The presented developments advance NMR of oriented samples for structure determination of membrane proteins and liquid crystals.