The progressive development of structure and stability during the equilibrium folding of the alpha subunit of tryptophan synthase from Escherichia coli
Pj. Gualfetti et al., The progressive development of structure and stability during the equilibrium folding of the alpha subunit of tryptophan synthase from Escherichia coli, PROTEIN SCI, 8(8), 1999, pp. 1623-1635
The urea-induced equilibrium unfolding of the alpha subunit of tryptophan s
ynthase (alpha TS), a single domain alpha/beta barrel protein, displays a s
table intermediate at similar to 3.2 M urea when monitored by absorbance an
d circular dichroism (CD) spectroscopy (Matthews CR, Crisanti MM, 1981, Bio
chemistry 20:784-792). The same experiment, monitored by one-dimensional pr
oton NMR, shows another cooperative process between 5 and 9 M urea that inv
olves His92 (Saab-Rincon G et al., 1993, Biochemistry 32:13981-13990). To f
urther test and quantify the implied four-state model, N <--(-->) I1 <--(--
>) I2 <--(-->) U, the urea-induced equilibrium unfolding process was follow
ed by tyrosine fluorescence total intensity, tyrosine fluorescence anisotro
py and far-UV CD. All three techniques resolve the four stable states, and
the transitions between them when the FL total intensity and CD spectroscop
y data were analyzed by the singular value decomposition method. Relative t
o U, the stabilities of the N, I1, and I2 states are 15.4, 9.4, and 4.9 kca
l mol(-1), respectively. I2 partially buries one or more of the seven tyros
ines with a noticeable restriction of their motion; it also recovers simila
r to 6% of the native CD signal. This intermediate, which is known to be st
abilized by the hydrophobic effect, appears to reflect the early coalescenc
e of nonpolar side chains without significant organization of the backbone.
I1 recovers an additional 43% of the CD signal, further sequesters tyrosin
e residues in nonpolar environments, and restricts their motion to an exten
t similar to N. The progressive development of a higher order structure as
the denaturant concentration decreases implies a monotonic contraction in t
he ensemble of conformations that represent the U, I2, I1, and N states of
alpha TS.