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-la beled 7,8-dihydroneopterin aldolase (DHNA) from Staphylococcus aureus, whic h is a symmetric homooctamer protein of molecular mass 110 kDa, showed 20-f old to 50-fold sensitivity gains when compared to the corresponding convent ional triple resonance NMR experiments. On this basis, sequential connectiv ities could be established for nearly all pairs of neighboring residues in DHNA. TROSY-type nuclear Overhauser enhancement spectroscopy yielded additi onal data to close the remaining gaps in the sequential assignment, and pro vided supplementary information on the secondary structure. Complete sequen ce-specific assignments of the 121-residue polypeptide chain in this 110 kD a octamer could thus be obtained in aqueous solution at 20 degrees C, and t he regular secondary structures in the solution conformation were found to coincide nearly identically with those in the crystal structure of the DHNA octamer.