The urea-induced equilibrium unfolding of the α
subunit of tryptophan synthase (αTS), a single domain
α/β barrel protein, displays a stable intermediate
at ∼3.2 M urea when monitored by absorbance and circular
dichroism (CD) spectroscopy (Matthews CR, Crisanti MM,
1981, Biochemistry 20:784–792). The same
experiment, monitored by one-dimensional proton NMR, shows
another cooperative process between 5 and 9 M urea that
involves His92 (Saab-Rincón G et al., 1993, Biochemistry
32:13981–13990). To further test and quantify
the implied four-state model, N [rlarr ] I1
[rlarr ] I2 [rlarr ] U, the urea-induced
equilibrium unfolding process was followed by tyrosine
fluorescence total intensity, tyrosine fluorescence anisotropy
and far-UV CD. All three techniques resolve the four stable
states, and the transitions between them when the FL total
intensity and CD spectroscopy data were analyzed by the
singular value decomposition method. Relative to U,
the stabilities of the N, I1, and I2
states are 15.4, 9.4, and 4.9 kcal mol−1,
respectively. I2 partially buries one or more
of the seven tyrosines with a noticeable restriction of
their motion; it also recovers ∼6% of the native CD
signal. This intermediate, which is known to be stabilized
by the hydrophobic effect, appears to reflect the early
coalescence of nonpolar side chains without significant
organization of the backbone. I1 recovers an additional
43% of the CD signal, further sequesters tyrosine residues
in nonpolar environments, and restricts their motion to
an extent similar to N. The progressive development
of a higher order structure as the denaturant concentration
decreases implies a monotonic contraction in the ensemble
of conformations that represent the U, I2,
I1, and N states of αTS.