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 be further divided into A-, B-, and C-subdomains. Previous studies showed that in vitro unfolding of firefly luciferase induced by guanidinium chloride can be described as a four-state equilibrium with two inactive intermediates (Herbst, R., et al. (1997) J. Biol. Chem. 272, 7099-7105). In order to monitor spectroscopically the conformational changes that occur in the different domains and subdomains during the multi-state unfolding process, we constructed a series of single-tryptophan mutants. These mutants were purified and characterized and shown to retain essentially all of the structural properties of the wild-type luciferase. Under equilibrium conditions, the unfolding of each mutant protein were studied by means of fluorescence and circular dichroism. The results show that different conformational changes occur in specific regions, suggesting a sequential unfolding process for firefly luciferase. Under 2.5 M GdmCl, whereas the N-domain unfolds partially holding half of the secondary structure content, the C-domain unfolds almost completely. In the equilibrium intermediate I(2), the secondary structure might stem mostly from the A- and B- subdomains.
Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai, 200031, People's Republic of China.
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D000818:Animals; D002942:Circular Dichroism; D001517:Coleoptera / Q000201:enzymology*; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D014364:Tryptophan; D008156:Luciferases