Multidimensional Heteronuclear Correlation Spectroscopy of a Uniformly 15N- and 13C-Labeled Peptide Crystal: Toward Spectral Resolution, Assignment, and Structure Determination of Oriented Molecules in Solid-State NMR

New one-, two-, and three-dimensional solid-state NMR spectroscopic methods designed for structural studies of uniformly 15 N- and 13 C-labeled peptides and proteins in oriented samples are described. These methods provide a means of obtaining resolved spectra, sequential resonance assignments, and structural constraints. Experimental results for model single-crystal peptides and amino acids demonstrate that high-resolution one-dimensional 13 C spectra can be obtained for signals from carbonyl or carboxyl ( 13 CO) carbons in uniformly labeled samples by applying phase-modulated selective homonuclear (PSH) decoupling at aliphatic carbon resonances, in addition to heteronuclear proton and 15 N decoupling. 13 C-detected two-dimensional 15 N/ 13 C chemical shift correlation spectroscopy is made possible by a combination of PSH decoupling and broadband heteronuclear polarization transfer sequences such as WALTZ-5 cross-polarization. Experimental two-dimensional spectra of uniformly 15 N- and 13 C-labeled AlaGlyGly crystals show that resolution and sequential assignment of 13 CO and 15 N NMR signals is possible. Comparisons of experimental spectra and simulations verify the assignments and the accuracy of structural information contained in the two-dimensional spectra in the form of the orientation-dependent 13 CO and 15 N chemical shifts. 13 C-detected three-dimensional spectroscopy is also demonstrated by adding a 1 H− 15 N dipolar dimension to the two-dimensional methods. Results of experiments at fields of 9.39 and 17.6 T (400 and 750 MHz proton NMR frequencies) are reported. Motivations for uniform labeling and 13 C detection in oriented systems and implications for future structural studies of oriented proteins are discussed.