Solution X-ray scattering analysis of cold- heat-, 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 (Durea). 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 Durea states. From Guinier plots of the scattering intensities, radii of gyration (Rg) were determined to be 20.1(+/- 1.8) A for N, and 20.7(+/- 1.3), 25.8(+/- 1.5) and 32 to 35 A for D', D and Durea, 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 Durea showed that the polypeptide chain has no globular part either in Durea or 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 Durea 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.
Konno
T
T
Division of Material Science, Graduate School of Science and Technology, Kobe University, Japan.
Kataoka
M
M
Kamatari
Y
Y
Kanaori
K
K
Nosaka
A
A
Akasaka
K
K
D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003080:Cold Temperature; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D011489:Protein Denaturation; D012542:Scattering, Radiation; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D012996:Solutions; D013302:Streptomyces / Q000737:chemistry*; D013381:Subtilisins / Q000037:antagonists & inhibitors; D014508:Urea / Q000737:chemistry*; D014965:X-Rays
D001426:Bacterial Proteins; D015842:Serine Proteinase Inhibitors; D012996:Solutions; C018150:subtilisin inhibitor protein, Streptomyces; D014508:Urea; D013381:Subtilisins