Triple-resonance NOESY-based experiments with improved spectral resolution: Applications to structural characterization of unfolded, partially folded and folded proteins

NMR-based structural studies of macromolecules focus to a large extent on the establishment of interproton distances within the molecule based on the nuclear Overhauser effect (NOE). Despite the improvements in resolution resulting from multidimensional NMR experiments, the detailed characterization of disordered slates of proteins or highly overlapped regions of folded molecules using current NMR methods remains challenging. A suite of triple-resonance NOESY-type pulse schemes is presented which require uniform N-15 and C-13 labeling and make use of the chemical shift dispersion of backbone N-15 and C-13' (carbonyl) resonances to increase the spectral resolution. In particular, for the case of partially folded and unfolded proteins, the experiments exploit the fact that the dispersion of N-15 and C-13' resonances is comparable to that observed in folded states. Ambiguities that arise in the assignment of NOEs as a result of the severe chemical shift degeneracy in H-1 and aliphatic C-13 nuclei are resolved, therefore, by recording the chemical shifts of N-15 or C-13' either before or after the NOE mixing period. Applications of these methods to the study of the unfolded state of the N-terminal SH3 domain of drk (drkN SH3) and a partially folded large fragment of staphylococcal nuclease (SNase), Delta 131 Delta, are presented. In addition, an application to folded SNase in complex with the ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+ is illustrated which allows the assignment of NOEs between degenerate H-alpha protons or protons resonating close to water.