Time-Resolved Small-Angle X-ray Scattering Study of the Folding Dynamics of Barnase

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 angstrom, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the Rg value extrapolated from kinetic R-g data to time zero, R-g,R-0, is 24.3 +/- 0.1 angstrom, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 angstrom). 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,D-cis) to be 25.7 +/- 0.6 angstrom. Moreover, R-g of the major component of Dphys containing the native proline isomer (D-phys,D-tra) was estimated as 23.9 +/- 0.2 angstrom based on R-g,R-0. Consequently, both components of the burst-phase intermediate of barnase (D-phys,D-tra and D-phys,D-cis) are still largely expanded. It was inferred that D-phys possesses the N-terminal helix and the center of the beta-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase. (C) 2010 Elsevier Ltd. All tights reserved.