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. 2022 Mar 21;8(4):473–482. doi: 10.1021/acscentsci.1c01577

Figure 5.

Figure 5

MicD-F and ArtGox act in tandem to install thiazoline and thiazole backbones within globular proteins. (A) Cartoons illustrating the sequences of RopC, RopM, and RopN and the conditions used for MicD-F-catalyzed cyclodehydration (left arrow) or tandem cyclodehydration/dehydrogenation catalyzed by MicD-F and ArtGox (right arrow). (B) Wavelength-dependent circular dichroism spectra of RopC, RopM, and RopN at [monomer] = 20 μM in 10 mM phosphate, 100 mM NaCl, 150 μM TCEP, pH 7.0 and 25 °C. (C) LC/MS analysis of the reaction of RopC, RopN, and RopM with MicD-F under the conditions shown in panel (A) above. The characteristic loss of 18 mass units upon cyclodehydration is evident for both RopC and RopM; the reaction of RopM was incomplete under these conditions. (D) Treatment of RopC with MicD-F and ArtGox under the conditions shown in panel (A) above led to clean conversion into the corresponding thiazole (RopC-Z). (E) The wavelength-dependent CD spectra of RopC-U and RopC-Z compared to RopC; these data are not corrected for contributions due to the thiazoline or thiazole linkage. (F) The melting temperatures of RopC, RopC-U, and RopC-Z (after refolding) are almost identical.