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 °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.