*2.6. Interaction of PldA-GFP IBs with Amyloid-Specific Dye Thioflavin T*

Recent studies have shown that recombinant protein in IBs can have, along with other conformations, the structure of an intermolecular β-sheet (cross-β conformation), which is characteristic of amyloid proto-fibrils and fibrils [36]. As is known, the bacterial outer membrane proteins with the β-pleated sheet secondary structure have a high propensity to form amyloid-like structures [37]. We have previously shown the presence of amyloids in PldA IBs formed in *E. coli* at 37 ◦C [33].

A specific fluorescent dye, Thioflavin T (ThT), was used for the detection of amyloids in PldA-GFP IBs [38]. It should be noted that ThT allows detecting the amyloids in IBs in the presence of a protein with the native β-sheet structure, i.e., to differentiate the β-sheet conformation of the native protein from the structure of an intermolecular β-sheet in an aggregated form (amyloid fibrils) of the same protein [39]. This is important in the present study as the native-like structure of PldA is a β-sheet.

The ThT fluorescence intensity at 484 nm increases by more than 100 times in the presence of PldA-GFP IBs suspended in PBS, which indicates the existence of amyloid-like structures in IBs. An increase in the incubation time of IBs in PBS leads to a slight increase in the dye fluorescence intensity (Figure 9).

**Figure 9.** Difference fluorescence spectra of Thioflavin T bound to PldA-GFP IBs. IBs were incubated in PBS (triangle) and 0.04% SDS (circle) for 2 h (solid symbols) and 24 h (empty symbols).

IBs treated with 0.04% SDS also bind ThT, which is accompanied by a 300-fold increase in the dye fluorescence intensity (Figure 9). The incubation time of IBs in a detergent solution (10 min and 24 h) does not affect the fluorescence intensity of a bound ThT. A three-fold enhancement in the fluorescence of ThT in the presence of IBs incubated in SDS relative to untreated IBs may be due to several reasons: an increase in both the content of amyloids in IBs and their availability for dye binding, or changes in the structural and physical characteristics of the amyloids. The supposed changes in the amyloids in detergent-treated IBs seem to be quite realistic, as IBs solubilization occurs in the presence of SDS, and it has also been shown that SDS can induce the formation and modification of amyloid-like structures [40,41].
