The progressive development of structure and stability during the equilibrium folding of the alpha subunit of tryptophan synthase from Escherichia coli

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.