title: Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase.
abstract: Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D(phys), and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R(g)) of the guanidine unfolded state (U) is 26.9±0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R(g) value extrapolated from kinetic R(g) data to time zero, R(g,0), is 24.3±0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in R(g)(2) was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R(g) of the minor component of D(phys) containing the non-native cis proline isomer (D(phys,cis)) to be 25.7±0.6 Å. Moreover, R(g) of the major component of D(phys) containing the native proline isomer (D(phys,tra)) was estimated as 23.9±0.2 Å based on R(g,0). Consequently, both components of the burst-phase intermediate of barnase (D(phys,tra) and D(phys,cis)) are still largely expanded. It was inferred that D(phys) possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.
authors.affiliation: Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Konuma
authors.forename: Tetsunari
authors.initials: T
authors.lastname: Kimura
authors.forename: Shuzo
authors.initials: S
authors.lastname: Matsumoto
authors.forename: Yuji
authors.initials: Y
authors.lastname: Goto
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Fujisawa
authors.forename: Alan R
authors.initials: AR
authors.lastname: Fersht
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takahashi
mesh_terms: D001426:Bacterial Proteins; D019791:Guanidine / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012260:Ribonucleases / Q000737:chemistry*; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D019791:Guanidine