Refolding of luciferase subunits from urea and assembly of the active heterodimer. Evidence for folding intermediates that precede and follow the dimerization step on the pathway to the active form of the enzyme.
Conditions have been established that allow reversible refolding of luciferase from 5 M urea. The kinetics of formation of the active enzyme showed a concentration-independent lag, suggesting the existence of intermediate structures on the pathway of refolding. The rate of approach to the final level of activity was strongly concentration-dependent at protein concentrations below 10 micrograms/ml, but at concentrations above about 20 micrograms/ml, the rate of approach to the final activity value did not change with concentration. The concentration dependence presumably reflects the second-order step yielding the heterodimeric structure. The finding that at concentrations above 20 micrograms/ml, the rate becomes insensitive to concentration suggests that under these conditions, some step subsequent to dimerization become rate-limiting. When the refolding reaction was initiated by dilution out of 5 M urea at 50 micrograms/ml followed at various times by a secondary dilution to a final concentration of 5 micrograms/ml, it was found that the increase in activity continued at the rate characteristic of the higher protein concentration for a period of about 1-2 min following the dilution before slowing to the rate expected for the lower protein concentration. These observations indicate that there are inactive heterodimeric species that form from assembly of the individual subunits and that these species must undergo further folding to yield the active heterodimeric species. At protein concentrations of 5-50 micrograms/ml, the final yield of active enzyme was about 65-85%, decreasing at higher and lower concentrations. At higher concentrations, aggregation probably accounts for the limit in recovery, whereas at lower concentrations, it appears that the reduced yield of activity is due to the competing process of the folding of one or both individual subunits into some form incompetent to interact with each other. These experiments demonstrate the existence of slow steps in the refolding of luciferase subunits from urea and the formation of the active heterodimeric structure, both preceding and following the dimerization. Furthermore, the failure of protein at low concentrations to efficiently reassemble into the active heterodimer is consistent with the prior finding that luciferase subunits produced independently in Escherichia coli fold into conformations that cannot interact to form the active heterodimer upin mixing (Waddle, J. J., Johnston, T. C., and Baldwin, T. O. (1987) Biochemistry 26, 4917-4921).
Center for Macromolecular Design, Texas A&M University, College Station 77843.
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Ziegler
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ME
Goldberg
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AF
Chaffotte
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TO
Baldwin
D003001:Cloning, Molecular; 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; D014508:Urea; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014508:Urea; D008156:Luciferases