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 states 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 ¹⁵N and ¹³C labeling and make use of the chemical shift dispersion of backbone ¹⁵N and ¹³C′ (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 ¹⁵N and ¹³C′ 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 and aliphatic ¹³C nuclei are resolved, therefore, by recording the chemical shifts of ¹⁵N or ¹³C′ 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), Δ131Δ, 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^α protons or protons resonating close to water.