Figure 2.

NMR verifies the disordered ORF6_CTR undergoes proline cis/trans isomerization. (A) 2D ¹H-¹⁵N HSQC spectrum of 300 μM ¹⁵N-labeled ORF6_CTR measured at 15°C, pH 6.9, and 14.1 T. Residues colored in green originate from the trans-P57 conformational ensemble, while residues colored in pink and labeled with an asterisk (^∗) report on the cis-P57 conformation. The black peak is unassigned. (B) 2D ¹H-¹³C strip taken from the 3D CC(CO)NH spectrum of the 300 μM ¹³C,¹⁵N-labeled ORF6_CTR at the ¹⁵N chemical shift of M58^∗. (C) The ORF6_CTR¹³C^α, ¹³C^β, and ¹H^α chemical shifts were used to calculate the secondary structure propensity scores for both the cis-P57 and trans-P57 subensembles. A positive value indicates α-helical propensity, a negative value indicates a β-strand propensity, whereas a value near zero indicates random coil. (D) The cis-P57 populations at 15°C (solid fill) and 37°C (dashed fill) are shown for each well-resolved cis-P57 and trans-P57 peak in the 2D ¹H-¹⁵N HSQC spectrum. The integrated peak volume was used to calculate the mean cis-P57 population and standard deviation across residues E55, Q56, and E59. (E) The van ’t Hoff analysis of P57 cis/trans isomerization determined from the integrated peak volume. The mean natural logarithm of the equilibrium constant for proline cis/trans isomerization about the Q56-P57 peptide bond as a function of temperature was calculated using residues E55, Q56, and E59. Error bars represent the standard deviation across the three residues used in the analysis. The van ’t Hoff linear fit yielded a cis-P57 population of 10 ± 2% at 37°C.