SAXS and CD spectra of αTS as a function of refolding time. (A) Kratky plots at 150 μs (dotted line), 1 ms (dashed line), and 5 ms (dotted-dashed line). The unfolded Kratky plot recorded at 6 M urea (solid line), normalized to the concentration used for the kinetic data, is shown for reference. The R_g of the native state of αTS is 18.1 Å (51). Refolding was initiated by a urea concentration dilution from 6 to 0.6 M. The final protein concentration was 1 mg·ml⁻¹. (Inset) Dependence of R_g on refolding time. Each scattering curve in A and time point in the Inset represents a total accumation time of ≈5 s. (B) CD spectra of αTS at various refolding times. Spectra at 150 μs (green open circles) and 5 ms (blue open circles), acquired after an 8–0.8 M urea concentration jump, are compared with the native spectrum (red open circles), the unfolded spectrum recorded at 8 M urea (black open circles) and the unfolded spectrum extrapolated to 0.8 M urea (black filled circles). The 150-μs spectrum, the native spectrum at 0.8 M urea, and the unfolded spectrum at 8 M urea were acquired with the same stock solutions in the continuous-flow mixer having a 127-μm optical path. The 5-ms time point spectrum was acquired by using the stopped-flow cuvette with a 1.5-mm optical path. Sample concentration was 60 μM for data recorded in the continuous-flow mixer and 10 μM for the stopped-flow data. The extrapolation of the unfolded baseline was performed by using published data and global fit parameters (52). Data were recorded at 21°C.

Gaussian distance distribution analysis of the time-resolved FRET data shown in Fig. 3. Results of global nonlinear least-squares analysis of tryptophan excited-state decays at various refolding time points using a double-Gaussian function are shown for the 15-212 (A) and 168-212 (B) samples. Distance distributions are shown at 53 (dark blue), 82 (light blue), 112 (green), 141 (yellow), and 170 (red) μs. Five pairs of donor-only and donor-acceptor-labeled decay traces were fit. The gray areas correspond to regions where the Trp–AEDEANS FRET pair is insensitive to distance changes. Based on the measured anisotropy of W212 (Fig. 5) and estimated anisotropy of AEDANS, values of κ² can range between 0.4 and 1.5, however, leading to an uncertainty in the distances of approximately ±10%. The Gaussians in A have peaks at 20 and 41 Å and corresponding full-width at half-maximum of 2.7 and 14.3 Å. The Gaussians in B have peaks at 16 and 46 Å and corresponding full-width at half-maximum of 21 and 39 Å, respectively.

Structure of αTS and location of chromophores used as FRET probes. X-ray crystal structure of αTS [PDB ID code:1BKS (49)] with FRET pairs 15-54, 15-134, 168-212 and 15-212 highlighted. The figure was prepared by using PyMol (50).

Average excited-state lifetimes of tryptophan as a function of refolding time following an 8 to 0.8 M urea concentration jump for four FRET pairs (green) and donor-only controls (red). (A) Pair 15-54. (B) Pair 15-134. (C) Pair 168-212. (D) Pair 15-212. (E–H) The corresponding FRET efficiencies are shown. The 〈EED〉_app calculated from the Förster equation are indicated on the right vertical axis. The uncertainty in the average lifetimes, determined from controls in which no kinetics are present, is ≈0.015 ns. A consistent set of kinetics is observed in the average Trp lifetimes and the FRET efficiencies, suggesting that the observed kinetics are not an artifact of the mutations or acceptor chromophore labeling. Data were recorded at 21°C.

Time-resolved anisotropy as a function of refolding time for F22W (A) and F212W (B) after an 8–0.8 M urea concentration jump. TCSPC curves were acquired every 10.93 μs in refolding time, and refolding times are indicated by the color-coding shown on the right. The total flow rate was 17.5 ml·min⁻¹ (1.75 ml·min⁻¹ protein and 15.75 ml·min⁻¹ buffer), which corresponds to a 32.65 μs·mm⁻¹ linear flow velocity in the channel. Data were recorded at 21°C in 10 mM phosphate buffer containing 0.2 mM K₂EDTA and 1 mM βME. Final protein concentration was 10 μM.