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The progressive development of structure and stability during the equilibrium folding of the α subunit of tryptophan synthase from Escherichia coli
Author(s) -
Gualfetti Peter J.,
Bilsel Osman,
Matthews C. Robert
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.8.1623
Subject(s) - tryptophan synthase , protein subunit , escherichia coli , folding (dsp implementation) , chemistry , protein folding , atp synthase , tryptophan , biochemistry , biophysics , biology , enzyme , amino acid , engineering , gene , electrical engineering
The urea‐induced equilibrium unfolding of the α subunit of tryptophan synthase (aTS), 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 52 :13981–13990). To further test and quantify the implied four‐state model, N ⇄ I1 ⇄ I 2 ⇄ 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, I 1, and I 2 states are 15.4, 9.4, and 4.9 kcal mol −1 , respectively. I 2 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. I 1 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, I 2, I 1, and N states of αTS.