*4.4. DENV C Recombinant Protein Production and Purification*

Recombinant DENV C protein expression and purification was conducted based on previous approaches [13]. We used a pET-21a plasmid containing DENV serotype 2 strain New Guinea C capsid protein gene (encoding amino acid residues 1–100) [69]. The protein was expressed in *Escherichia coli* C41 and C43 bacteria grown in lysogeny broth (LB) medium. The only differences in the purification protocol are the abolition of the ammonium sulfate precipitation step and the addition of a size exclusion chromatography step (with Sephadex S200) after the heparin affinity column chromatography, using an AKTA chromatography equipment. The C protein was purified in a 55 mM KH2PO4, pH 6.0, 550 mM KCl. DENV C protein purified fractions were concentrated with Amicon Ultra-4 Centrifugal Filters of 3 or 10 kDa nominal cut-off, from Millipore (Billerica, MA, USA). Concentrated protein samples were stored at −80 ◦C. Protein samples quality was assessed by SDS-PAGE and matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Very low degradation and the highest peak consistent with the expected mass of the protein monomer (11765 Da).

#### *4.5. Time-Resolved Fluorescence Anisotropy*

Time-resolved fluorescence spectroscopy measurements were performed in a Life Spec II equipment with an EPLED-280 pulsed excitation light-emitting diode (LED) of 275 nm (Edinburgh Instruments, Livingston, UK), acquiring the emission at 350 nm. DENV C (monomer) concentration was 20 μM in 50 mM KH2PO4, 200 mM KCl, pH 6.0 or pH 7.5, with 550 μL total volume, in 0.5 cm × 0.5 cm quartz cuvettes. The instrument response function, IRF(*t*), was obtained with the same settings, except emission, which was at 280 nm, with a solution of polylatex beads of 60 nm diameter diluted in Mili-Q water. Measurements were performed at 22 ◦C. Time-resolved fluorescence intensity measurements with picosecond-resolution were obtained by the time-correlated single-photon timing (TCSPT) methodology [35]. Measurements were performed at constant time, with 15 min per decay, acquiring 2048 time points in a 50 ns window. Four intensity decays, *I*(*t*), were acquired in each condition, with excitation/emission polarizers, respectively at vertical/vertical positions, *I*VV(*t*), vertical/horizontal

positions, *I*VH(*t*), horizontal/vertical positions, *I*HV(*t*), and horizontal/horizontal positions, *I*HH(*t*). The instrumental *G*-factor was calculated as [35]:

$$G = \frac{\int\_0^{50} I\_{\rm HV}(t)dt}{\int\_0^{50} I\_{\rm HH}(t)dt} \tag{1}$$

The *G*-factor value obtained was 1.61. The intensity decay with emission polarizer at the magic angle (~54.7◦, with respect to the vertical excitation polarizer), *Im*(*t*), avoids the effects of anisotropy. It can be calculated easily [35]:

$$I\_m(t) = l\_{\rm VV}(t) + 2G l\_{\rm VH}(t) \tag{2}$$

with *I*VV(*t*) and *I*VH(*t*) depending on the time-resolved fluorescence anisotropy, *r*(*t*), as:

$$I\_{\rm VV}(t) = \frac{I\_m(t)}{3}(1 + 2r(t))\tag{3}$$

$$I\_{\rm VH}(t) = \frac{I\_m(t)}{\Re G} (1 - r(t)) \tag{4}$$

Thus, *Im*(*t*) was used to obtain the fluorescence lifetime components, τ*i*, and the respective amplitudes, α*i*, for the DENV C W69. *Im*(*t*) was described by a sum of three exponential terms:

$$I\_{\text{ll}}(t) = \sum\_{i=1}^{3} \alpha\_i e^{\left(-\frac{t}{t\_i}\right)} \tag{5}$$

where the index *i* represents each component of the fluorescence decay. For the fitting to the data, α*<sup>i</sup>* and τ*<sup>i</sup>* values were obtained by iteratively convoluting *Im*(*t*) with the IRF(*t*):

$$I\_m^{\text{calc}}(t) = I\_m(t) \otimes \text{IRF}(t) \tag{6}$$

and fitting *I calc <sup>m</sup>* (*t*) to the experimental data, *I exp <sup>m</sup>* (*t*), using a non-linear least squares regression method. The usual statistical criteria, namely a reduced χ<sup>2</sup> value bellow 1.3 and a random distribution of weighted residuals, were used to evaluate the goodness of the fits [35]. Data analysis was performed using the TRFA Data Processing Package v1.4 (Scientific Software Technologies Centre, Belarusian State University, Minsk, Belarus) which allows calculating automatically the standard error (SE) for each fitted parameter [35].

The time-resolved fluorescence anisotropy, *r*(*t*), is calculated via *I*VV(*t*), *I*VH(*t*) and *G* via\_ENREF\_52:

$$r(t) = \frac{I\_{\rm VV}(t) - GI\_{\rm VH}(t)}{I\_{\rm VV}(t) + 2GI\_{\rm VH}(t)} \tag{7}$$

In this case, the obtained *r*(*t*) can be fitted to a single exponential decay [35]:

$$r(t) = r\_0 e^{(-\frac{t}{\tau\_c})} \tag{8}$$

where *r*<sup>0</sup> is the anisotropy when *t*→0 and τ*<sup>c</sup>* is the rotational correlation time. The *r*(*t*) decays were globally analyzed in TRFA Data Processing Package v1.4 maintaining the previously obtained α<sup>i</sup> and τ*<sup>i</sup>* values constant, and convoluting Equations (3) and (4) with the respective IRF(*t*), analogously to the analysis of *Im*(*t*), using Equation (8) to fit *r*(*t*). Values obtained for both pH conditions were considered statistically different if their 95% confidence intervals (~1.96 × SE) do not overlap (corresponding to *p* < 0.05).
