Apparent radii of the native, stable intermediates and unfolded conformers of the α-subunit of tryptophan synthase from E. coli, a TIM barrel protein

The urea-induced equilibrium unfolding of the α-subunit of tryptophan synthase (αTS) from Escherichia coli can be described by a four-state model, N ⇆ I1 ⇆ I2 ⇆ U, involving two highly populated intermediates, I1 and I2 [Gualfetti, P.J., Bilsel, O., and Matthews, C.R. (1999) Protein Sci. 8, 1623- 1635]. To extend the physical characterization of these stable forms, the apparent radius was measured by several techniques. size-exclusion chromatography (SEC), analytical ultracentrifugation (UC), and dynamic light scattering (DLS) experiments yield an apparent Stokes radius, R(s), of ~24 Å for the native state of αTS. The small-angle X-ray scattering (SAXS) experiment yields a radius of gyration, R(g), of 19.1 Å, consistent with the value predicted from the X-ray structure and the Stokes radius. As the equilibrium is shifted to favor I1 at ~3.2 M and I2 at 5.0 M urea, SEC and UC show that R(s) increases from ~38 to ~52 Å. Measurements of the radius by DLS and SAXS between 2 and 4.5 M urea were complicated by the self- association of the I1 species at the relatively high concentrations required by those techniques. Above 6 M urea, SEC and UC reveal that R(s) increases linearly with increasing urea concentration to ~54 Å at 8 M urea. The measurements of R(s) by DLS and R(g) by SAXS are sufficiently imprecise that both values appear to be identical to the I2 and U states and, considering the errors, are in good agreement with the results from SEC and UC. Thermodynamic parameters extracted from the SEC data for the N ⇆ I1 and I1 ⇆ I2 transitions agree with those from the optical data, showing that this technique accurately monitors a part of the equilibrium model. The lack of sensitivity to the I2 ⇆ U transition, beyond a simple swelling of both species with increasing urea concentration, implies that the Stokes radii for the I2 and U states are not distinguishable. Surprisingly, the hydrophobic core known to stabilize I2 at 5.0 M urea [Saab-Rincon, G., Gualfetti, P.J., and Matthews, C.R. (1996) Biochemistry 35, 1988-1994] develops without a significant contraction of the polypeptide, i.e., beyond that experienced by the unfolded form at decreasing urea concentrations. Kratky plots of the SAXS data, however, reveal that I2, similar to N and I1, has a globular structure while U has a more random coil-like form. By contrast, the formation of substantial secondary structure and the burial of aromatic side chains in I1 and, eventually, N are accompanied by substantial decreases in their Stokes radii and, presumably, the size of their respectively conformational ensembles.