Three-dimensional 13C shift/1H-15N Coupling/15N shift solid-state NMR correlation spectroscopy

Triple-resonance experiments capable of correlating directly bonded and proximate carbon and nitrogen backbone sites of uniformly C-13- and N-15-labeled peptides in stationary oriented samples are described. The pulse sequences integrate cross-polarization from H-1 to C-13 and from C-13 to N-15 With flip-flop (phase and frequency switched) Lee-Goldburg irradiation for both C-13 homonuclear decoupling and H-1-N-15 spin exchange at the magic angle. Because heteronuclear decoupling is applied throughout, the three-dimensional pulse sequence yields C-13 shift/H-1-N-15 coupling/N-15 shift correlation spectra with single-line resonances in all three frequency dimensions. Not only do the three-dimensional spectra correlate C-13- and N-15 resonances, they are well resolved due to the three independent frequency dimensions, and they can provide up to four orientationally dependent frequencies as input for structure determination. These experiments have the potential to make sequential backbone resonance assignments in uniformly C-13- and N-15-labeled proteins. (C) 1999 Academic Press.