Fig. 2: Investigation of the molecular mechanism and general applicability of SIMTeGES. | Nature Communications

Fig. 2: Investigation of the molecular mechanism and general applicability of SIMTeGES.

From: Intein-mediated temperature control for complete biosynthesis of sanguinarine and its halogenated derivatives in yeast

Fig. 2

a Quantification of conformational strain on the backbone involves assessing the deviation from the peptide bond planarity near the scissile peptide bond. Gray spheres represent C atoms, blue spheres represent N atoms, and red spheres represent O atoms. b Measurement of the average planarity of the peptide bond in GAL4-tsINT after achieving molecular dynamic equilibrium, recorded at both 30 °C and 25 °C. c SIMTeGES for temperature-controlled expression of GAL80, employing the colored metabolite lycopene as a reporter system. d SIMTeGES for temperature-dependent expression of mCherry. e Western blot analysis of mCherry-INT, mCherry-dINT, and mCherry-tsINT, validating the direct correlation between host protein activity and intein splicing. f Western blot analysis of the splicing kinetics of mCherry-tsINT. The unspliced protein is ~85 kDa, while the spliced form is around 35 kDa. Blots were representative of at least two independent experiments (2e and 2 f). Significance was calculated using two-way ANOVA followed by Tukey’s multiple comparisons test. Data are presented as mean ± s.d. (n  =  3 biologically independent samples). Source data are provided as a Source Data file.

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