Solution X-ray Scattering Analysis of Cold- Heat-, and Urea-denatured States in a Protein, StreptomycesSubtilisin Inhibitor

Abstract

Streptomycessubtilisin 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 ureastates. From Guinier plots of the scattering intensities, radii of gyration (R g) were determined to be 20.1(±1.8) Å for N, and 20.7(±1.3), 25.8(±1.5) and 32 to 35 Å 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 ureashowed that the polypeptide chain has no globular part either in D ureaor D. Combined with the results from circular dichroism and 1H 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 ureait 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 part with a unique tertiary structure and a part disordered and expanded to a degree comparable to D. f2 f2 Abbreviations used: SSI, Streptomycessubtilisin inhibitor; N, native state of SSI; D′, cold-denatured state of SSI; D, heat-denatured state of SSI; D urea, urea-denatured state of SSI-;SAXS, small-angle X-ray scattering; R g, radius of gyration; CD, circular dichroism; NMR, nuclear magnetic resonance; PGK, yeast 3-phosphoglycerate kinase, MW, molecular mass; ppm, parts per million.