SOLUTION X-RAY-SCATTERING ANALYSIS OF COLD-DENATURED, HEAT-DENATURED, AND UREA-DENATURED STATES IN A PROTEIN, STREPTOMYCES SUBTILISIN INHIBITOR

Streptomyces subtilisin inhibitor (SSI), a homo-dimeric protein with a subunit of 113 residues with two disulfide bonds, is known to exist at low pH in at least three distinct thermodynamic states namely, the native (N), cold-denatured (D') and heat-denatured (D). Small-angle X-ray scattering was used to analyze and to compare overall chain conformations of SSI in typical, N, D', D and urea-denatured states (D-urea). Molecular masses were determined from scattering intensities extrapolated to a scattering angle of zero, which showed that SSI exists as a homo-dimer in the N state, but as dissociated monomers in the D', D and D-urea states. From Guinier plots of the scattering intensities, radii of gyration (R(g)) were determined to be 20.1(+/-1.8) Angstrom for N, and 20.7(+/-1.3), 25.8(+/-1.5) and 32 to 35 Angstrom for D', D and D-urea, respectively Kratky plots for both N and D' exhibited a bell-shape indicating that the polypeptide chain has a globular part not only in N but also in D', while Kratky plots for D and D-urea showed that the polypeptide chain has no globular part either in D-urea or D. Combined with the results from circular dichroism and H-1 NMR spectra, a picture emerges for the polypeptide chain conformation of SSI such that in N it is a globular dimer close to that in the crystal, in D-urea it is totally disordered and expanded nearly to a fully random chain with restrictions only from the disulfide bridges, in D the entire chain is disordered and expanded but with considerable local intra-chain interactions, and in D' the chain consists of a part with a unique tertiary structure and a part disordered and expanded to a degree comparable to D. (C) 1995 Academic Press Limited