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

Triple-resonance experiments capable of correlating directly bonded and pro ximate carbon and nitrogen backbone sites of uniformly C-13- and N-15-label ed peptides in stationary oriented samples are described. The pulse sequenc es 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-dimensio nal pulse sequence yields C-13 shift/H-1-N-15 coupling/N-15 shift correlati on spectra with single-line resonances in all three frequency dimensions. N ot only do the three-dimensional spectra correlate C-13- and N-15 resonance s, 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.