Volume 112, Issue 12, Pages 2540-2551 (June 2017) Direct Conversion of an Enzyme from Native-like to Amyloid-like Aggregates within Inclusion Bodies  Francesco Elia, Francesca Cantini, Fabrizio Chiti, Christopher Martin Dobson, Francesco Bemporad  Biophysical Journal  Volume 112, Issue 12, Pages 2540-2551 (June 2017) DOI: 10.1016/j.bpj.2017.05.011 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 (A) Sso AcP variants investigated in this study: WT (black); L65A (red); ΔN11 (gray); and ΔN11 L65A (orange). The edge β-strand 4 and residue at position 65 are labeled. The same colors are used in the subsequent figures. (B) Fraction of the folded state for the four variants investigated in this study as a function of GndHCl concentration, measured through equilibrium unfolding experiments. The data for ΔN11 L65A (orange) were measured in this study and those for WT (black), L65A (red), and ΔN11 (gray) are reproduced from previous studies (28,31,32). The solid lines represent best fits to a two-state model (33); the thermodynamic data obtained from these fits are reported in the text. To see this figure in color, go online. Biophysical Journal 2017 112, 2540-2551DOI: (10.1016/j.bpj.2017.05.011) Copyright © 2017 Biophysical Society Terms and Conditions

FIgure 2 [15N,1H] HSQC spectra of the four variants investigated in this study recorded in the lysis buffer at 700 MHz after protein expression and cell lysis. Data are shown for WT (black), L65A (red), ΔN11 (gray), and ΔN11 L65A (orange). To see this figure in color, go online. Biophysical Journal 2017 112, 2540-2551DOI: (10.1016/j.bpj.2017.05.011) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Deposition of Sso AcP in IBs. (A) Representative SDS-PAGE bands for the Sso AcP variants present in IBs extracted from E. coli cells at the indicated times after induction of protein expression. (B) Relative amounts of Sso AcP variants present in IBs extracted from E. coli cells, evaluated using a densitometric analysis of the SDS-PAGE bands, as a function of time after induction of protein expression. The data on the y axis correspond to the band intensities of the different variants normalized to the sum of all band intensities present in the IBs and resulting from all proteins (n = 5; error bars represent the mean ± SE). To see this figure in color, go online. Biophysical Journal 2017 112, 2540-2551DOI: (10.1016/j.bpj.2017.05.011) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 Assessment of the relative content of native-like and amyloid-like conformations of the Sso AcP variant in IBs (n = 5; error bars represent the mean ± SE). (A) Enzymatic activity in the presence of IBs extracted from bacterial cells, expressed as the percentage of the activity measured in the tested IB compared to that of an identical amount of folded WT Sso AcP, as a function of time after induction of protein expression. The insets show the reaction catalyzed by Sso AcP (left) and the absorption spectra (right) of the reactants (benzoylphosphate) and products (benzoate and phosphate) at equimolar concentrations. (B) ThT fluorescence in the presence of IBs extracted from bacterial cells, normalized to the amount of protein precipitated in the IBs after subtraction of the ThT signal recorded in the presence of identical amounts of insoluble proteins extracted from E. coli cells not expressing Sso AcP, as a function of time after induction of protein expression. In both (A) and (B), data are shown for WT (black), ΔN11 (gray), L65A (red), and ΔN11 L65A (orange). The data corresponding to 3.0 and 6.0 h are highlighted in green and cyan, respectively. To see this figure in color, go online. Biophysical Journal 2017 112, 2540-2551DOI: (10.1016/j.bpj.2017.05.011) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 Structural investigation of Sso AcP present in IBs. (A and E) CR raw absorption spectra measured in the presence of IBs extracted from bacterial cells expressing Sso AcP after 3.0 h (A) and 6.0 h (E) of expression. The band at 550 nm is highlighted in yellow. Dashed lines represent the scattering of IBs in the absence of CR. The spectrum of CR in the absence of IBs (CR alone) is depicted in cyan. (B and F) Differences between the CR spectra measured in the presence and absence of IBs, as shown in (A) and (E), after subtraction of IB scattering and normalization to the amount of Sso AcP in the IB. (C and G) FTIR spectra of IBs extracted from bacterial cells expressing Sso AcP after 3.0 h (C) and 6.0 h (G) of expression. The band located at 1620–1630 cm−1 is highlighted in yellow. (D and H) Second-order derivatives of the spectra shown in (E) and (F). In (A)–(H), data are shown for WT (black), ΔN11 (gray), L65A (red), and ΔN11 L65A (orange). To see this figure in color, go online. Biophysical Journal 2017 112, 2540-2551DOI: (10.1016/j.bpj.2017.05.011) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 A model for the incorporation of Sso AcP into IBs and its subsequent conversion into an amyloid-like entity in E. coli cells (red and green arrows), shown in comparison with the pathway followed by other systems (blue arrows). After translation and folding (black arrow), proteins commonly follow a set of possible pathways that involve misfolding and formation of amyloid-like assemblies, which only eventually are incorporated into IBs (blue arrows). In contrast, in the case of Sso AcP, the protein becomes incorporated into IBs as a native-like molecule (first phase; red arrow), which later converts into highly organized amyloid-like aggregate (second phase; green arrow). To see this figure in color, go online. Biophysical Journal 2017 112, 2540-2551DOI: (10.1016/j.bpj.2017.05.011) Copyright © 2017 Biophysical Society Terms and Conditions