The structural characteristics of Azotobacter vinelandii apoflavodoxin II have been determined using multidimensional NMR spectroscopy. Apoflavodoxin has a stable, well-ordered core but its flavin binding region is flexible. The local stability of apoflavodoxin was probed using hydrogen/deuterium exchange measurements. The existence of an apoflavodoxin equilibrium folding intermediate is inferred from the non-coincidence of CD and fluorescence unfolding curves obtained for the guanidinium hydrochloride induced unfolding of apoflavodoxin. We suggest that the structured part of the putative intermediate is composed of the elements of secondary structure which have the slowest exchanging amide protons in the native protein. These elements are strands beta1, beta3, beta4 and beta5a and helices alpha4 and alpha5. We propose that it is a general feature of flavodoxins that the stable nucleus resides in the C-terminal part of these proteins. The results on flavodoxin are compared with those on two sequentially unrelated proteins sharing the flavodoxin-like fold: Che Y and cutinase. It is shown that the stable nucleus is found in different parts of the flavodoxin-like topology.
Copyright 1998 Academic Press.