THE EFFECT OF THE METAL-ION ON THE FOLDING ENERGETICS OF AZURIN, A COMPARISON OF THE NATIVE, ZINC AND APOPROTEIN

The unfolding by guanidine hydrochloride (GuHCl) and the refolding on dilution of zinc and apoazurin have been monitored by far-UV circular dichroism (CD). With the native protein, the unfolding was followed by CD and optical absorption in the visible spectral region. With the zi nc protein, the reversible unfolding has also been followed by tryptop han fluorescence and NMR. The zinc and Cu1+ metal ions remain associat ed with the protein in the unfolded state. When the unfolding of the n ative protein is followed by CD, the initial, reversible transition du e to unfolding is followed by a slow change associated with the reduct ion of Cu2+ by the thiol group of the ligand Cys112. The unfolding of apoazurin displays two CD transitions, which evidence suggests represe nt different folding domains, the least stable one including the metal -binding site and the other one the rest of the beta-sheet structure. Both occur at a lower GuHCl concentration than the unfolding of the na tive protein. The CD titrations also demonstrate that zinc azurin has a lower stability than the copper protein, Unfolding of zinc azurin fo llowed by tryptophan fluorescence occurs at a much lower GuHCl concent ration than the CD changes, and NMR spectra show that there is no loss of secondary and tertiary structure at this concentration, whereas th e CD-detected loss of secondary structure correlates with the NMR chan ges. Thus, the fluorescence change is ascribed to a small local pertur bation of the structure around the single tryptophan residue. The diff erences in stability of the three forms of azurin are discussed in ter ms of the rack mechanism. A bound metal ion stabilizes the native fold , and this stabilization is larger for Cu(II) than for Zn(II), reflect ing the higher affinity of the protein for Cu(II). (C) 1997 Elsevier S cience B.V.