Nature research paper: NMR-guided directed evolution
The activity of 0.1 µM AlleyCat10 was tested with 0.12-0.96 mM substrate in 20 mM HEPES, 100 mM NaCl, pH 7.0 at 22 °C with 10 mM CaCl or 100 mM EDTA . Data are presented as the mean values and the error bars represent standard deviations obtained from three independent measurements.
Spectra for the proteins , 3.1 µM for FerrElCat) were collected in 20 mM HEPES, pH 7.0 in an anaerobic cuvette with a 1 cm pathlength at room temperature. Extended Data Fig. 7 Michaelis-Menten plots of Kemp elimination catalyzed by reduced myoglobin mutants and by AlleyCat proteins. Final reaction mixtures for myoglobin mutant analyses contained 1 mM L-ascorbic acid, 0.1 µM SOD, 20 nM catalase, 140-840 µM substrate, 1.5% acetonitrile in 20 mM Tris . The protein concentration was 1 µM for Mb, 0.1 or 0.25 µM for Mb-based double variants, 5 nM for Mb or Mb-based double or triple variants. For the AlleyCat proteins reaction mixtures contained 0.1 µM proteins with 0.12-0.96 mM substrate in 1.5% acetonitrile, 20 mM Tris, pH 8.0, 10 mM CaCl
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