NMR assignment and secondary structure determination of an octameric 110 kDa protein using TROSY in triple resonance experiments

TROSY-type triple resonance experiments with the uniformly H-2,C-13,N-15-labeled 7,8-dihydroneopterin aldolase (DHNA) from Staphylococcus aureus, which is a symmetric homooctamer protein of molecular mass 110 kDa, showed 20-fold to 50-fold sensitivity gains when compared to the corresponding conventional triple resonance NMR experiments. On this basis, sequential connectivities could be established for nearly all pairs of neighboring residues in DHNA. TROSY-type nuclear Overhauser enhancement spectroscopy yielded additional data to close the remaining gaps in the sequential assignment, and provided supplementary information on the secondary structure. Complete sequence-specific assignments of the 121-residue polypeptide chain in this 110 kDa octamer could thus be obtained in aqueous solution at 20 degrees C, and the regular secondary structures in the solution conformation were found to coincide nearly identically with those in the crystal structure of the DHNA octamer.