Protein folding and stability investigated by fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy: the flavodoxin story

In this review, the experimental results obtained on the folding and stabil ity of Azotobacter vinelandii flavodoxin are summarised. By doing so, three main spectroscopic techniques used to investigate protein folding and stab ility are briefly introduced. These techniques are: circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, and nuclear magnetic res onance (NMR) spectroscopy in combination with the hydrogen exchange methodo logy. Results on the denaturant-induced and thermal equilibrium unfolding o f apoflavodoxin from A. vinelandii, i.e. flavodoxin in the absence of the r iboflavin-5'-monophosphate (FMN) cofactor, are discussed. A scheme for the equilibrium unfolding of apoflavodoxin is presented which involves a relati vely stable molten globule-like intermediate. Denaturant-induced apoflavodo xin (un)folding as followed at the residue-level by NMR shows that the tran sition of native A. vinelandii apoflavodoxin to its molten globule state is highly co-operative. However, the unfolding of the molten globule to the u nfolded state of the protein is non-co-operative. A comparison of the foldi ng of A. vinelandii flavodoxin with the folding of flavodoxin from Anabaena PCC 7119 is made. The local stabilities of apo- and holoflavodoxin from A. vinelandii as measured by NMR spectroscopy are compared. Both Che Y and cu tinase, which have no sequence homology with apoflavodoxin but which share the flavodoxin-like topology, have stabilisation centres different from tha t of apoflavodoxin from A. vinelandii. The stable centres of structurally s imilar proteins can thus reside in different parts of the same protein topo logy. Insight in the variations in (local) unfolding processes of structura lly similar proteins can be used to stabilise proteins with a flavodoxin-li ke fold. Finally, the importance of some recent experimental and theoretica l developments for the study of flavodoxin folding is briefly discussed. (C ) 2000 Elsevier Science B.V. All rights reserved.