FIGURE 2.

NMR evidence that ISCU exists in solution as two slow interchanging conformational states and that the S ⇄ D equilibrium is perturbed by single amino acid substitutions. A, ¹H,¹⁵N HSQC NMR spectrum of ISCU. Because ISCU contains only one tryptophan (Trp-76), the presence of two cross-peaks in the boxed region and inset indicates the existence of two different conformational states. Assignments of individual peaks to the S- and D-states are indicated. B, ¹H,¹⁵N HSQC NMR spectra of ISCU variants with shifted S ⇄ D equilibria. Whereas the substitutions D39V and N90A stabilize the S-state, substitutions D39A and H105A stabilize the D-state. C, expansions of the spectra in B show the Trp-76 side chain signals used to quantify the relative populations of the S- and D-states. All NMR spectra were collected at 600 MHz (¹H) at 25 °C with solutions at pH 8.0.