On the nature of conformational openings: Native and unfolded-state hydrogen and thiol-disulfide exchange studies of ferric aquomyoglobin

Native-state amide hydrogen exchange (HX) of proteins in the presence of de naturant has provided valuable details on the structures of equilibrium fol ding intermediates. Here, we extend HX theory to model thiol group exchange (SX) in single cysteine-containing variants of sperm whale ferric aquomyog lobin. SX is complementary to HX in that it monitors conformational opening events that expose side-chains, rather than the main chain, to solvent. A simple two-process model, consisting of EX2-limited local structural fluctu ations and EX1-limited global unfolding, adequately accounts for all HX dat a. SX is described by the same model except at very low denaturant concentr ations and when the bulky labeling reagent 5,5'-dithiobis (2-nitrobenzoic a cid) (DTNB) is used. Under these conditions SX can occur by a novel denatur ant-dependent process. This anomalous behavior is not observed when the sma ller labeling reagent methyl methanethiosulfonate is employed, suggesting t hat it reflects a denaturant-induced increase in the amplitudes of local st ructural fluctuations. It also is not seen in heme-free apomyoglobin, which may indicate that local openings are sufficiently large in the absence of denaturant to allow DTNB unhindered access. Differences in SX kinetics obta ined using the two labeling reagents provide estimates of the sizes of loca l opening reactions at different sites in the protein. At all sequence posi tions examined except for position 73, the same opening event appears to fa cilitate exchange of both backbone amide and side-chain thiol groups. The C 73 thiol group is exposed by a low-energy fluctuation that does not expose its amide group to exchange. (C) 2001 Academic Press.