Probing local conformational changes during equilibrium unfolding of firefly luciferase: Fluorescence and circular dichroism studies of single tryptophan mutants

Firefly luciferase is a monomeric protein composed of two globular domains. There is a wide cleft between the two domains. The N-terminal domain can b e further divided into A-, B-, and C-subdomains. Previous studies showed th at in vitro unfolding of firefly luciferase induced by guanidinium chloride can be described as a four-state equilibrium with two inactive intermediat es (Herbst, R., et al. (1997) J. Biol. Chem. 272, 7099-7105). In order to m onitor spectroscopically the conformational changes that occur in the diffe rent domains and subdomains during the multi-state unfolding process, we co nstructed a series of single-tryptophan mutants. These mutants were purifie d and characterized and shown to retain essentially all of the structural p roperties of the wild-type luciferase. Under equilibrium conditions, the un folding of each mutant protein were studied by means of fluorescence and ci rcular dichroism, The results show that different conformational changes oc cur in specific regions, suggesting a sequential unfolding process for fire fly luciferase. Under 2.5 M GdmCl, whereas the N-domain unfolds partially h olding half of the secondary structure content, the C-domain unfolds almost completely. In the equilibrium intermediate I-2, the secondary structure m ight stem mostly from the A- and B-subdomains. (C) 2001 Academic Press.