STRUCTURAL CHARACTERIZATION OF APOFLAVODOXIN SHOWS THAT THE LOCATION OF THE STABLE NUCLEUS DIFFERS AMONG PROTEINS WITH A FLAVODOXIN-LIKE TOPOLOGY

The structural characteristics of Azotobacter vinelandii apoflavodoxin II have been determined using multidimensional NMR spectroscopy. Apof lavodoxin has a stable, well-ordered core but its flavin binding regio n is flexible. The local stability of apoflavodoxin was probed using h ydrogen/deuterium exchange measurements. The existence of an apoflavod oxin equilibrium folding intermediate is inferred from the noncoincide nce of CD and fluorescence unfolding curves obtained for the guanidini um hydrochloride induced unfolding of apoflavodoxin. We suggest that t he structured part of the putative intermediate is composed of the ele ments of secondary structure which have the slowest exchanging amide p rotons in the native protein. These elements are strands beta 1, beta 3, beta 4 and beta 5 alpha and helices alpha 4 and alpha 5. We propose that it is a general feature of flavodoxins that the stable nucleus r esides in the C-terminal part of these proteins. The results on flavod oxin are compared with those on two sequentially unrelated proteins sh aring the flavodoxin-like fold: Che Y and cutinase. It is shown that t he stable nucleus is found in different parts of the flavodoxin-like t opology. (C) 1998 Academic Press.