Figure 6. ACCuRET reveals the NTD and CTD likely do not form a complex at rest.

(A) Normalized FRET efficiency between L-ANAP on the NTD and Cu²⁺-TETAC at position C469 at pH 8 (black) and pH 6 (red). FRET efficiencies, SEM, N and calculated distances are summarized in Table 1. Zn²⁺-TETAC negative control for positions E8 and G11 shows that the changes in signal measured with Cu²⁺-TETAC are due the presence of Cu²⁺ and not due to any other changes. (B) Normalized FRET efficiency between L-ANAP on the NTD and Cu²⁺-TETAC at position C477 at pH 8 (black) and pH 6 (red). The inset cartoons show the position of L-ANAP denoted by a red star and the single cysteine denoted by a green circle. Box and whisker plot with whiskers ranging from minimum to maximum values and the bottom and top edges of the box denoting the 25^th and 75^th quartiles, respectively. Statistical significance shown using two-way ANOVA with Tukey’s multiple comparisons test are denoted between relevant comparison using appropriate asterisks. Ns indicates p > 0.05, * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, **** indicates p ≤ 0.0001.