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. 2019 Aug 8;9:11409. doi: 10.1038/s41598-019-47657-9

Figure 2.

Figure 2

Ensilication improves the thermal stability of Ag85b. (a) Thermal melt circular dichroism (CD) spectra showing irreversible thermal unfolding of Ag85b. CD spectra at 5 °C, 20 °C, 85 °C and upon return to 20 °C are displayed in the inset figure. Deconvolution was performed using DichroWeb. (b) Boltzmann sigmoidal fit of CD thermal denaturation curve at 222 nm depicting a melting temperature of 53.9 °C. (c) Temperature-induced loss of Ag85b structural integrity visualised by SDS-PAGE. (d) Ag85b ensilication trials for visualisation of sol-gel precipitates showing 50 mM glycine at pH 7.4 yields the highest precipitation. Water was included as a negative control. (e) SDS-PAGE analysis illustrating the composition of the released solution and unensilicated supernatant following ensilication of Ag85b in the glycine buffer or presence of Sbi III-IV. See Supplementary Table S2 for protein concentrations. (f) CD spectroscopy results depicting secondary structural comparability of native and released Ag85b. Spectra are representative of 3 replicates. Deconvolution was performed using DichroWeb. (g) SDS-PAGE illustrating thermal degradation and aggregation of native but not released Ag85b. Particle analysis and CD of thermally-treated native and released Ag85b can be found in Supplementary Figs S2 and S3 respectively. Full-length gel images of (c,e,g) are included in Supplementary Figs S14–S16.