Figure 5.

Analysis of φ_ex ¹³C^α and methyl ¹³C spins in HP67. Values ${\phi }_{ex}^{1/2}$ determined from individual fits of R_ex and the global value of k_ex are plotted versus |Ω_sec| for (a) ¹³C^α (●, ○) and (b) methyl ¹³C (■, □) spins in HP67. Solid symbols in (a) and (b) represent data for residues that are assumed to be fully unfolded in the intermediate ensemble; in each plot, the solid line depicts the linear least-squares fit of these data points. In (a) the fit is computed using only ¹³C^α data and the line has a slope of 0.101 ± 0.007 and therefore an estimated population of the intermediate of (1.01 ± 0.14)%. Similarly, in (b) the fit of the methyl ¹³C data yields a slope of 0.114 ± 0.007 and therefore an estimated population of the intermediate of (1.30 ± 0.16)%. In (c) the data from both molecular probes are combined and fit collectively for points colored green. The global fit of the fully disordered residues, shown as a dashed green line, yields a slope of 0.104 ± 0.005, corresponding to a population of (1.09 ± 0.11)% for intermediate. The solid line has a slope of 0.0105, corresponding to p₂ = (1.11 ± 0.09)% observed previously for ¹⁵N spin relaxation dispersion. Data points that were excluded from the fit are grouped into two categories: (i) those that lie above and to the left of the fitted are presumed to adopt non-native conformations in the intermediate and (ii) those that lie below and to the right of the fit are presumed to maintain residual native-like interactions in the intermediate. Residues in (i) and (ii) are colored with brown and blue gradients, respectively, with the color of the data points becoming lighter as the ratio |Δδ/Ω_sec| deviates from unity, in which Δδ was obtained from φ_ex assuming p₂ = (1.11 ± 0.09)%.