*4.7. DLS*

DLS measurements were performed at 25 ◦C in 100 mM phosphate buffer (pH 8.0) with 500 mM NaCl at different protein concentrations. Experiments were performed at fixed angle (θ = 173◦) in a Zetasizer nano instrument (Malvern Instrument Ltd., Malvern, UK) equipped with a 10-mW helium-neon laser (λ = 632.8 nm) and a thermoelectric temperature controller. Experiments were analyzed with Zetasizer software (Malvern Instrument Ltd., Malvern, UK), and they were carried out as described [50]. Each sample was measured 10 times with 10 runs of 30 s each. The Z-average size was obtained by fitting the autocorrelation function with the cumulants method. The *R*<sup>S</sup> was calculated by applying the Stokes–Einstein equation: *D = kT/*6πη*R*S, where *k* is the Boltzmann constant and *T* is the temperature (in K). Experiments were also carried out at pH 4.5 (100 mM acetic acid) and 500 mM NaCl, but the poor signal obtained precluded any reliable conclusion.

#### *4.8. Molecular Modelling*

Disorder propensity was estimated by using scoring functions according to independent algorithms [26–29], as calculated through submission to their respective web servers. Three-dimensional models of the protein were obtained using the on-line structure predictors I-TASSER [33], FALCON [34], SWISS-MODEL [35], and Robetta [36]. Folding stability in the LrtA structure predicted by I-TASSER was assessed in MD simulations at room temperature performed with the GROMACS package [54]. The AMBER ff99SB-ILDN force field [55] and TIP3P model [56] were used for the protein and water, respectively, and other simulation conditions were as previously

described [57,58]. High temperature simulations were performed at 400 and 500 K, according to a protocol previously adopted [59].
