Equilibrium intermediates in the reversible unfolding of firefly (Photinus pyralis) luciferase.

Firefly luciferase has been used as a model protein to study cotranslational and chaperone-assisted protein folding. We found conditions for reversible unfolding of luciferase in the absence of cellular factors, and we characterized denaturant-induced equilibrium unfolding transitions and refolding kinetics of the enzyme. Luciferase unfolding induced by guanidinium chloride at 10 degrees C can be described as a four-state equilibrium with two inactive intermediates highly populated around 1 and 3 M denaturant. The transitions occur around 0.3, 1.7, and 3.8 M denaturant. The free energy of denaturation to the first inactive intermediate (DeltaG0N <==> I1 = 15 +/- 3 kJ.mol-1) is small for a protein of 60 kDa. Fluorescence and circular dichroism spectra of the intermediates indicate that I1 has a compact conformation, whereas aromatic side chains are highly exposed in the second intermediate, I2, despite its high content of secondary structure. In the presence of a hydrophilic detergent, significant reactivation of luciferase is observed up to temperatures at which the native protein is unstable. Reactivation kinetics of luciferase are exceedingly slow and probably not limited by proline isomerization, as suggested by their independence from the time spent in the unfolded state.