Apoflavodoxin (un)folding followed at the residue level by NMR

The denaturant-induced (un) folding of apoflavodoxin from Azotobacter vinel andii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of H-1-N-15 he teronuclear single-quantum coherence spectra recorded at increasing concent rations of guanidinium hydrochloride despite the formation of protein aggre gate. These NH groups are distributed throughout the whole apoflavodoxin st ructure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy, Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. T he NMR (un)folding data support a mechanism for apoflavodoxin folding in wh ich a relatively stable intermediate is involved. Native apoflavodoxin is s hown to cooperatively unfold to a molten globule-like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR ch emical shift timescale. The subsequent unfolding of the molten globule is f aster on the NMR chemical shift timescale and the limited appearance of H-1 -N-15 HSQC cross peaks of unfolded apoflavodoxin in the denaturant range st udied indicates that it is noncooperative.