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 bas ed on the nuclear Overhauser effect (NOE). Despite the improvements in resolution resulting from multidimensional NMR experiments, the detai led characterization of disordered slates of proteins or highly overla pped regions of folded molecules using current NMR methods remains cha llenging. A suite of triple-resonance NOESY-type pulse schemes is pres ented which require uniform N-15 and C-13 labeling and make use of til e chemical shift dispersion of backbone N-15 and C-13' (carbonyl) reso nances to increase the spectral resolution. In particular, for the cas e of partially folded and unfolded proteins, the experiments exploit t he fact that the dispersion of N-15 and C-13' resonances is comparable to that observed in folded states. Ambiguities that arise in the assi gnment of NOEs as a result of the severe chemical shift degeneracy in H-1 and aliphatic C-13 nuclei are resolved, therefore, by recording th e chemical shifts of N-15 or C-13' either before or after the NOE mixi ng 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 f olded large fragment of staphylococcal nuclease (SNase), Delta 131 Del ta, are presented. In addition, an application to folded SNase in comp lex with the ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+ is i llustrated which allows the assignment of NOEs between degenerate H-al pha protons or protons resonating close to water.