Multidimensional heteronuclear correlation spectroscopy of a uniformly N-15- and C-13-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 N-15- and C-13-labeled peptid es 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 pepti des and amino acids demonstrate that high-resolution one-dimensional C-13 s pectra can be obtained for signals from carbonyl or carboxyl ((CO)-C-13) ca rbons in uniformly labeled samples by applying phase-modulated selective ho monuclear (PSH) decoupling at aliphatic carbon resonances, in addition to h eteronuclear proton and N-15 decoupling. C-13-detected two-dimensional N-15 /C-13 chemical shift correlation spectroscopy is made possible by a combina tion of PSH decoupling and broadband heteronuclear polarization transfer se quences such as WALTZ-5 cross-polarization. Experimental two-dimensional sp ectra of uniformly N-15- and C-13-labeled AlaGlyGly crystals show that reso lution and sequential assignment of (CO)-C-13 and N-15 NMR signals is possi ble. Comparisons of experimental spectra and simulations verify the assignm ents and the accuracy of structural information contained in the two-dimens ional spectra in the form of the orientation-dependent (CO)-C-13 and N-15 c hemical shifts. C-13-detected three-dimensional spectroscopy is also demons trated by adding a H-1-N-15 dipolar dimension to the two-dimensional method s. Results of experiments at fields of 9.39 and 17.6 T (400 and 750 MHz pro ton NMR frequencies) are reported. Motivations for uniform labeling and C-1 3 detection in oriented systems and implications for future structural stud ies of oriented proteins are discussed.