Figure 2.

Measurement of ¹⁵N chemical shifts during VA2-ubiquitin folding. (A, B) Small region of the HSQC spectrum, recorded with the scheme of Figure 1 for τ = 10 ms and κ = 2 ms, modulated by (A) cos(ω_Nκ) and (B) sin(ω_Nκ). Extension of the 50-ms t₁ time domain to 75 ms by the NUS reconstruction program SMILE³⁴ was used to slightly increase both resolution and sensitivity. Negative intensity is shown in brown. For full spectra, see Figure S3. (C) M_x(τ) and (D) M_y(τ) magnetization of residue S57 after evolution for κ = 0.5 ms (red) and κ = 2 ms (blue), and (E) the corresponding magnitude |M(τ)|, with these values normalized to |M(τ)| = 1 at τ = 248 ms. Solid colored lines correspond to the values predicted for a two-state folding model with ρ = 14.3 s⁻¹ and known chemical shifts¹⁴ of the unfolded (F, dashed line) and folded (F, dotted) states at 1 bar. (F) Apparent average chemical shift <ω_N/2π> at time τ after the pressure drop. (G-J) Analogous to (C-F) but for residue R72 with solid lines corresponding to a three-state model with k_u→i = 13.2 s⁻¹; k_u→f = 6.5 s⁻¹; and k_i→f = 12.0 s⁻¹, and colored dashed lines for the two-state model. The band at 126.6 ppm in (J) depicts the fitted ¹⁵N shift of the intermediate state. The full set of residues is shown in Figure S4. The error bars in (C-E) and (G-I) indicate the RMS noise in the M_x and M_y spectra, scaled by the normalization factor of |M|. Error bars in (F) and (J) indicate the uncertainty in the average chemical shift derived from the RMS noise in the M_x and M_y spectra