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. Author manuscript; available in PMC: 2020 Dec 29.
Published in final edited form as: J Am Chem Soc. 2020 Apr 10;142(16):7245–7249. doi: 10.1021/jacs.9b11520

Figure 2.

Figure 2.

Selection of Tet-v3.0 RSs and characterization of Tet-v3.0 reactivity. (A) Tet-v3.0 amino acids with similar reactivity to Tet-v2.0 (B) Tet-v2.0Me (green) clashes with RS backbone whereas Tet-v3.0Me is eliminates backbone clash. (C) Measuring fluorescence of expressed GFP-Tet-v3.0 showed high efficiency and broad permissivity when other Tet-v3.0 amino acids were supplemented at 0.4 mM. (D) ESI-Q-TOF mass spectrometry confirmed Tet-v3.0Me incorporation into GFP and quantitative labeling with sTCO. (GFP-wt predicted: 27827.02 Da avg, observed 27827.49 Da avg; GFP-Tet-v3.0Me predicted: 27954.17 Da avg, observed: 27954.54 Da avg; GFP-Tet-v3.0Me+sTCO predicted: 28078.39 Da avg, observed: 28078.61 Da avg). The lower mass peak labeled with * is a loss of n-terminal methionine and higher mass peaks are salt sodium and potassium adducts. (E) Measured second order rate constants of free Tet-v3.0 and GFP-Tet-v3.0 with sTCO in PBS, pH 7.4 (Fig S3 and S7).