A stable intermediate in the equilibrium unfolding of Escherichia coli citrate synthase.
Urea-induced unfolding of Escherichia coli citrate synthase occurs in two phases, as monitored by circular dichroism at 222 nm (measuring secondary structure) or by tryptophan fluorescence. In this paper we characterize the intermediate state, which retains about 40% of the ellipticity of the native state, and is stable between 2.5 M and 5.5 M urea, approximately. This intermediate binds significant amounts of the probe for hydrophobic surfaces, anilinonaphthalene sulfonate, but forms aggregates at least as high as an octamer, as shown by transverse urea gradient polyacrylamide electrophoresis. Thermal denaturation of E. coli citrate synthase also produces an intermediate at temperatures near 60 degrees C, which also retains about 40% of the native ellipticity and forms aggregates, as measured by electrospray-ionization/time-of-flight mass spectrometry. We have used a collection of "cavity-forming" mutant proteins, in which bulky buried hydrophobic residues are replaced by alanines, to explore the nature of the intermediate state further. A certain amount of these mutant proteins shows a destabilized intermediate, as measured by the urea concentration range in which the intermediate is observed. These mutants are found in parts of the citrate synthase sequence that, in a native state, form helices G, M, N, Q, R, and S. From this and other evidence, it is argued that the intermediate state is an aggregated state in which these six helices, or parts of them, remain folded, and that formation of this intermediate is also likely to be a key step in the folding of E. coli citrate synthase.
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Department of Chemistry, University of Manitoba, Winnipeg, Canada.
Duckworth
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D002950:Citrate (si)-Synthase / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D014508:Urea; D002950:Citrate (si)-Synthase