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.