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Y454S mutation is thermally less stable relative to WT HARS. 10 μM enzyme (WT black circles, Y454S red triangles and R362H blue squares) was incubated with Sypro Orange dye in the absence (filled symbols) of substrate, or presence of 5 mM histidine (empty symbols). A) Wild type ± histidine. B) Wild type vs. Y454S. C) Y454S ± histidine. D) R362H ± histidine. Thermal shift results are from two preparations of WT and Y454S enzymes each measured in triplicate.
