Contribution of folding steps involving the individual subunits of bacterial luciferase to the assembly of the active heterodimeric enzyme.
Bacterial luciferase is an alpha beta heterodimer with a single active center in which the reaction of reduced FMN, O2, and an aliphatic aldehyde yields a photon of blue-green light. We have shown that refolding of the luciferase subunits from 5 M urea occurs via the intermediacy of several species, one of which is an inactive heterodimeric structure, resulting from the dimerization of alpha and beta, which isomerizes to the active alpha beta structure in a first-order reaction (Ziegler, M. M., Goldberg, M. E., Chaffotte, A. F., and Baldwin, T. O. (1993) J. Biol. Chem. 268, 10760-10765). We have also demonstrated the existence of an inactive heterodimeric species that is well populated at equilibrium in the presence of 1.6-2.8 M urea (Clark, A. C., Sinclair, J. F., and Baldwin, T. O. (1993) J. Biol. Chem. 268, 10773-10779). We have separated the alpha and beta subunits by ion exchange chromatography and investigated the effects on reformation of active luciferase of allowing the individual subunits to refold separately prior to mixing. These investigations show that the lag in formation of active luciferase is due to slow steps in folding of the individual subunits. The beta subunit appears to fold faster than the alpha subunit, but folding of the beta subunit also shows a distinct lag. When the alpha and beta subunits were allowed to refold from urea for periods of several hours or more prior to mixing, the yield of active heterodimeric luciferase was compromised, which is consistent with the finding that individual subunits produced in vivo fold into structures incompetent to interact with each other to form the active heterodimer (Waddle, J. J., Johnston, T. C., and Baldwin, T. O. (1987) Biochemistry 26, 4917-4921). It appeared that the rate with which the beta subunit assumed the heterodimerization-incompetent structure was faster than the rate with which the alpha subunit became heterodimerization-incompetent. These observations support a model for folding and assembly of the subunits of luciferase in which the two subunits fold into assembly-competent structures that associate to form the heterodimer. In a slow competing process, the subunits undergo a conformational rearrangement to form stable structures incompetent to form heterodimers. It appears that the association of the luciferase subunits might constitute an example of one polypeptide modifying the folding pathway of another, a model that is consistent with the suggestion that the formation of the heterodimeric structure of luciferase is a kinetic trap on the folding pathway of the individual subunits (Sugihara, J., and Baldwin, T. O. (1988) Biochemistry 27, 2872-2880).
Center for Macromolecular Design, Texas A&M University, College Station 77843.
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Baldwin
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Ziegler
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Chaffotte
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Goldberg
D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008954:Models, Biological; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D008156:Luciferases