Sequential unfolding of adenylate kinase during denaturation by guanidine hydrochloride

The unfolding of adenylate kinase in GuHCl of increasing concentrations has been followed by a combination of different methods. Molecular packing was measured by size-exclusion chromatography (SEC), exposure of buried Tyr residues by second- derivative spectra, loss of secondary structure by circular dichroism in the far-ultraviolet and the decrease in surface hydrophobicity by ANS binding. The conformational changes of adenylate kinase as followed by the above methods depend differently on GuHCl concentration. The concentrations of GuHCl at which 50% changes as measured by the above four methods occur are 0.3, 0.46, 0.64 and 0.64 M, respectively. SEC measurements show that with increasing GuHCl concentrations, the process of unfolding of adenylate kinase involves two slowly interconvertible intermediate stages, I1, and I2, the last is in a more advanced state of unfolding but is still more compact than the fully unfolded state, U, as indicated by their elution volumes in the SEC profile. There is also evidence to suggest that both the intermediates I1 and I2 may contain additional intermediary components in rapid equilibrium as indicated by the gradual shift of both peaks in the SEC elution profile. A sequential mechanism is suggested for the unfolding of adenylate kinase with increasing guanidine hydrochloride concentrations.