*2.4. Analysis of Dengue Virus (DENV) C Protein Rotational Correlation Time*

Given the close similarities between *Flavivirus* C proteins (Figures 1–4), DENV C can be used as a general model for them. Hence, we proceeded to determine DENV C overall rotational correlation time (τ*c*), taking advantage of the tryptophan residue in position 69 (W69) intrinsic fluorescence. Our computational data support three main structure/dynamics regions, including a disordered N-terminal region, which would increase its expected apparent size (as it would not be globular and folded), a property detectable by such an approach. Upon testing molecules in aqueous solution and at room temperature, fluorescence lifetimes are usually in the ns timescale, and the fluorescence decays are sensitive to the anisotropy of the fluorophore, which depends on its τ*<sup>c</sup>* (vd. Equations (1)–(8), describing

these relations, in the Methods section [34,35]). Thus, the time-resolved fluorescence decay of DENV C W69 and the corresponding anisotropy decay were determined, both at pH 6.0 and 7.5 (Figure 5).

**Table 2.** Fitting parameters of DENV C time-resolved fluorescence anisotropy data analysis. Parameters obtained from fitting Equations (5) and (8) to the data of Figure 5. Values are average (±% standard error, SE). \* Statistically significant differences (*p* < 0.05) between the values obtained for the two pH values tested.


**Figure 5.** DENV C time-resolved fluorescence anisotropy. Time-resolved fluorescence decay at pH (**a**) 6.0 and (**c**) 7.5, with the corresponding anisotropy decays at pH (**b**) 6.0 and (**d**) 7.5. Fluorescence and anisotropy decays at both pH values are similar (gray and black decays, respectively). Fitting of experimental data (red) took into account the instrument response function (IRF; in green) and the corresponding residuals distribution, displayed below each graph. The equations used for fitting are presented on the Methods Equations (5) and (8). The parameters obtained are shown in Table 2.

Time-resolved fluorescence anisotropy decays at both pH values are similar (Figure 5b,d). Fluorescence lifetime components (τ1, τ<sup>2</sup> and τ3) were obtained from the intensity decays Equations (2)–(6) [34,35], with a triple-exponential retrieving the best fit (Figure 5a,c). Fitting the data retrieves similar values Table 2 for τ1, τ<sup>2</sup> and τ3, and corresponding weights (α1, α<sup>2</sup> and α<sup>3</sup> pre-exponential factors, respectively). For accurate calculation of τ*c*, the condition τ*<sup>c</sup>* < 3 × τ<sup>3</sup> must occur [34,35]. Since τ<sup>3</sup> values were ~6.4 ns (with a significant weight α<sup>3</sup> of ~0.42), this means that, at both pH values, we could measure τ*<sup>c</sup>* values up to a limit of ~19 ns. In both pH conditions, the τ*<sup>c</sup>* measured was

16.4 ± 0.5 ns at 22 ◦C, within the limit and higher than expected for a purely globular protein of DENV C size, as predicted [13].

Rossi et al. [36] correlated the τ*<sup>c</sup>* of 16 globular proteins at 20 ◦C with their molecular weight (MW in kDa), based on NMR data, leading to the relation: τ*<sup>c</sup>* ≈ 0.6 MW. Assuming DENV C as a 23.5 kDa fully globular homodimer and correcting for the temperature (*T*) and viscosity (η) [37], the τ*<sup>c</sup>* predicted is 12.0 ns. However, the correlational time must be slightly higher, as the protein will be partially unfolded and disordered (in the N-terminal). Jones et al. [16] measured a τ*<sup>c</sup>* of 13 ns at 27 ◦C, by NMR, which with the corrections from Equation (10) [37], corresponds to 13.4 ns at 25 ◦C. Given DENV C size, this implies that the protein is not globular, in line with current knowledge of DENV C structure and dynamics [12–16]. Fluorescence anisotropy supports an even more open and partially disordered DENV C structure, given the τ*<sup>c</sup>* value of 15.2 ± 0.5 ns at 25 ◦C Table 3, in line with in silico data (Figures 1–4).

**Table 3.** Comparing DENV C τ*<sup>c</sup>* values (τ*<sup>c</sup>* at 25 ◦C in H2O were calculated using Equation (10)).

