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

The urea-induced equilibrium unfolding of the cr-subunit of tryptophan synt hase (alpha TS) from Escherichia coli can be described by a four-state mode l, N reversible arrow I1 reversible arrow 12 reversible arrow U, involving two highly populated intermediates, I1 and 12 [Gualfetti, P. J., Bilsel, O. , and Matthews, C. R. (1999) Protein Sci. 8, 1623-1635]. To extend the phys ical characterization of these stable forms, the apparent radius was measur ed by several techniques. Size-exclusion chromatography (SEC), analytical u ltracentrifugation (UC), and dynamic light scattering (DLS) experiments yie ld an apparent Stokes radius, Rs, of similar to 24 A for the native state o f alpha TS. The small-angle X-ray scattering (SAXS) experiment yields a rad ius of gyration, R-g, of 19.1 Angstrom, consistent with the value predicted from the X-ray structure and the Stokes radius. As the equilibrium is shif ted to favor I1 at similar to 3.2 M and 12 at 5.0 M urea, SEC and UC show t hat Rs increases from similar to 38 to similar to 52 Angstrom. Measurements of the radius by DLS and SAXS between 2 and 4.5 M urea were complicated by the self-association of the II species at the relatively high concentratio ns required by those techniques. Above 6 M urea, SEC and UC reveal that R-s increases linearly with increasing urea concentration to similar to 54 Ang strom at 8 M urea. The measurements of R-s by DLS and R, by SAXS are suffic iently imprecise that both values appear to be identical for the I2 and U s tates and, considering the errors, are in good agreement with the results f rom SEC and UC. Thermodynamic parameters extracted from the SEC data for th e N reversible arrow I1 and I1 reversible arrow I2 transitions agree with t hose from the optical data, showing that this technique accurately monitors a part of the equilibrium model. The lack of sensitivity to the 12 reversi ble arrow U transition, beyond a simple swelling of both species with incre asing urea concentration, implies that the Stokes radii for the 12 and U st ates are not distinguishable. Surprisingly, the hydrophobic core known to s tabilize 12 at 5.0 M urea [Saab-Rincon, G., Gualfetti, P. J., and Matthews, C. R. (1996) Biochemistry 35, 1988-1994] develops without a significant co ntraction of the polypeptide, i.e., beyond that experienced by the unfolded form at decreasing urea concentrations. Kratky plots of the SAXS data, how ever, reveal that 12, similar to N and Il, has a globular structure while U has a more random coil-like form. By contrast, the formation of substantia l secondary structure and the burial of aromatic side chains in Il and, eve ntually, N are accompanied by substantial decreases in their Stokes radii a nd, presumably, the size of their respective conformational ensembles.